Methods for treating or preventing cancer in a kras-variant patient and for diagnosing risk of developing multiple primary breast tumors

ABSTRACT

The invention relates to methods for preventing cancer in a KRAS-variant subject which include administering to the KRAS-variant subject an amount of estrogen effective to reduce the risk of developing cancer. In another aspect, the invention further relates to methods for treating cancer in a KRAS-variant subject, which include gradually decreasing estrogen exposure in the KRAS-variant subject to reduce the risk of aggressive tumor growth. In another aspect, the invention relates to a method of predicting an increased risk of developing a second, independent breast cancer in a subject. The method can include detecting a single nucleotide polymorphism (SNP) at position 4 of the let-7 complementary site 6 of KRAS in a patient sample wherein the presence of said SNP indicates an increased risk of developing a second, independent cancer in said subject.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. ProvisionalApplication No. 62/091,357, filed Dec. 12, 2014, the entire contents ofwhich are hereby incorporated by reference.

BACKGROUND

The KRAS-variant is a biologically functional, microRNA binding sitevariant in the KRAS oncogene, which predicts increased cancer riskespecially for women. MicroRNA (miRNA) binding site variants in the 3′untranslated region (3′UTR) of important growth and survival genes are arecently discovered novel class of germ-line mutations, which arepowerful biomarkers of cancer risk and treatment response (Cipollini etal. (2014) PHARMACOGENETICS AND PERSONALIZED MEDICINE 7:173-191).

One of the first mutations discovered in this class is the KRAS-variant,a let-7 binding site mutation in the 3′UTR of the KRAS oncogene (Chin etal. (2008) CANCER RES 68:8535-40). This mutation predicts an increasedrisk of several cancers, including non-small cell lung cancer (Id.),triple negative breast cancer (TNBC) in premenopausal women (Paranjapeet al. (2011) THE LANCET ONCOLOGY 12(4):377-386) and ovarian cancer(Ratner et al. (2010) CANCER RESEARCH 15:6509-15; Ratner et al. (2012)ONCOGENE 31(42):4559-66; Pilarski et al. (2012) PLOS ONE 7(5):e37891).The KRAS-variant has also been shown to predict unique tumor biology,with tumors in KRAS-variant patients exhibiting a KRAS-addictedsignature as well as an estrogen-negative, basal-like gene expressionpattern (Rather, 2012, supra; Paranjape, supra). Women with theKRAS-variant have also been found to be at a significantly increasedrisk of developing multiple primary cancers, including breast andovarian cancer, as well as a third independent cancer in the sameindividual (Pilarski, supra).

Women with the KRAS-variant are also at a significantly increased riskof developing multiple primary cancers, including breast and ovariancancer, as well as a third independent cancer in their lifetime(Pilarski, supra). Multiple primary cancer, although difficult topredict, is not rare, as up to one in eight cancer patients will bediagnosed with a new primary cancer after their first cancer diagnosis(metachronous cancer), and one in forty patients will be diagnosed withtwo cancers at the same time (synchronous cancer) (Levi et al. (2014)EUR J CANCER PREY doi: 10.1097/CEJ.0000000000000085). While it ishypothesized that metachronous cancers may be caused by primary cancertreatment, it is also thought that genetics plays a significant role inthe development of both synchronous and metachronous cancers (Bhatia(2014) CANCER. doi: 10.1002/cncr.29096; Amer (2014) CANCER MANAG RES5:119-34). Multiple primary breast cancer (MPBC) is one of the mostcommon forms of multiple primary cancer (Howe et al. (2005) BREASTCANCER RES TREAT 90(3):223-232), yet it remains difficult to identifythose at risk. Currently identified risk factors for the development ofmultiple or bilateral primary breast cancers include young age at firstdiagnosis (Raymond et al. (2006) BRITISH JOURNAL OF CANCER 94(11):1745-1750; Marcu et al. (2014) EUROPEAN JOURNAL OF CANCER CARE23(1):51-64; Kurian et al. (2009) JOURNAL OF THE NATIONAL CANCERINSTITUTE 101(15):1058-1065); first BC of lobular histology (Howe,supra; Chen et al. (1999) CANCER EPIDEMIOL BIOMARKERS PREV 8(10):855-61;Narod (2014) NATURE REVIEWS CLINICAL ONCOLOGY 11 (3): 157-166); high BMI(>30) in pre-menopausal patients with a hormone-receptor negative firstprimary (Brooks et al. (2012) BREAST CANCER RES TREAT 131(2):571-580);positive family history of breast cancer (Reiner et al. (2013) JOURNALOF CLINICAL ONCOLOGY 31(4):433-439); and mutations in BRCA 1, BRCA 2(Malone et al. (2010) JOURNAL OF CLINICAL ONCOLOGY 28(14):2404-2410;Metcalfe et al. (2011) BRITISH JOURNAL OF CANCER 104(9):1384-1392) orCHEK 2 (Broeks et al. (2004) BREAST CANCER RES TREAT 83(1):91-3).Additionally, in a small case series, the KRAS-variant was found in57.1% of uninformative (BRCA negative) patients who developed bilateralbreast cancer and ovarian cancer (Pilarski, supra). Factors decreasingmultiple primary breast cancer risk have also been identified, andinclude menarche after 13 years of age, multiparity (Narod, supra),treatment with anti-hormonal agents or chemotherapy (Clarke et al.(2005) LANCET 365(9472):1687-717; Alkner et al. (2009) EUR J CANCER45(14):2496-502) and prophylactic surgical intervention (Metcalfe,supra; Lostumbo et al. (2004) COCHRANE DATABASE SYST REV (4):Cd002748).These findings indicate that second BC risk can be impacted by estrogenalterations either before or after the first BC diagnosis.

Evidence that estrogen exposure increases primary breast cancer riskincludes increased BC risk in women experiencing early menarche, latemenopause, obesity, nulliparity or advanced maternal age at first birth(Anderson et al. (2014) BREAST CANCER RESEARCH AND TREATMENT144(1):1-10). In addition, in vitro studies using the breast epithelialline MCF10A support the hypothesis that excess estrogen and itsmetabolites can lead to increased transformation, or breast cancerinitiation (Liu et al. (2004) BREAST JOURNAL 10(6):514-521; Wang et al.(2013) ONCOGENE 32(44):5233-5240). However, it appears that estrogen isnot a risk for breast cancer for all women, as has come to light throughdata surrounding hormone replacement therapy use (HRT) (Beral (2003)LANCET 362(9382):419-427; Chlebowski et al. (2013) JOURNAL OF THENATIONAL CANCER INSTITUTE 105(8):526-535). Initially, the Million WomenStudy and Women's Health Initiative reported that current and/orprolonged use of HRT correlated with an increased risk of breast cancer.Because these tumors tended to be lower grade, with over-representationof lobular or tubular subtypes compared to other ductal cancers (Calleet al. (2009) CANCER 115(5):936-45), it was hypothesized that HRT wascausing cancers that otherwise would not have arisen. However, afollow-up WHI report found that there was actually no increased breastcancer risk for patients assigned to estrogen-only preparations comparedto placebo (Anderson et al. (2004) JAMA 291(14):1701-12). In fact, aftera median follow up of 11.8 years (IQR 9.1-12.9), post-menopausal use ofestrogen alone was associated with a lower breast cancer incidence thanplacebo (HR 0.77 (CI 0.62-0.95, p 0.02)) (Id.).

For women with the KRAS-variant, there is growing evidence that estrogenmay differentially impact their overall cancer risk and tumor biology.This includes: a higher risk of non-small cell lung cancer for womenversus men with the ERAS-variant (unpublished data); ovarian cancer riskalmost exclusively in post-menopausal women with the KRAS-variant(Pilarski, supra); in increased risk of estrogen receptor (ER) negativetumor development in KRAS-variant patients (TNBC (Paranjape, supra) andtype II uterine cancer (Lee et al. (2014) PLos ONE, 9(4):e94167), and;evidence that KRAS-variant post-menopausal breast cancer patients with ahistory of HRT are significantly more likely to develop biologicallyaggressive breast cancer (Cerne et al. (2012) BMC CANCER 12(105)). Thesefindings suggest that estrogen alterations may differentially impact theKRAS-variant, and, there is in fact strong evidence that such miRNAbinding site mutations are “influenced” by external exposure (Salzman etal. (2011) NAT MED 17:934-5). This is believed to be through alterationsin miRNAs, which are the immediate responders to cellular stress, andwhich directly act through the 3′UTR sites affected by these mutations(Id.).

Substantial evidence that the KRAS-variant acts as a cancer biomarker ofresponse to therapy also exists. This includes cisplatin resistance inKRAS-variant patients with ovarian or head and neck cancer (Ratner,2012, supra; Chung et al. (2014) ANN ONCOL, July 31. [Epub ahead ofprinting]), cetuximab sensitivity in ERAS-variant patients with coloncancer (Saridaki et al. (2014) CLIN CANCER RES 20(17):4499-510) or headand neck cancer (Chung, supra), and erlotonib resistance but sorafenibsensitivity in KRAS-variant patients with non-small cell lung cancer(NSCLC)(Weidhaas et al. (2014) J CLIN ONCOL 32(52):suppl; abstr 8135).Cell line data further supports the unique response of the KRAS-variantto chemotherapy exposures (Saridaki, supra).

Accordingly, there is a need in the art for methods to prevent and treatcancer in subjects with KRAS-variant. In addition, there is a need inthe art for methods to predict who is at risk for the development ofsecond primary breast tumors, so that preventative measures can be takenand treatment appropriately administered.

SUMMARY OF THE INVENTION

The present invention relates to the discovery that for women with theKRAS-variant, estrogen withdrawal appears to increase risk for bothtumor development as well as aggressive breast cancer tumor biology,both in vivo and in vitro. The present invention relates to the furtherdiscovery that KRAS-variant breast cancer patients are at asignificantly elevated risk for both synchronous and asynchronous secondbreast cancer development, which is not otherwise explained by otherknown risk factors.

Accordingly, the present invention relates to methods for preventingcancer in a KRAS-variant subject which include administering to theKRAS-variant subject an amount of estrogen effective to reduce the riskof developing cancer. For example, the method may include administeringto a KRAS-variant subject a tapering dose of estrogen, starting with thepatient's pre-oophorectomy baseline estrogen levels, or pre-chemotherapyestrogen levels, which can be calculated. In certain embodiments, themethod may include administering HRT to an oophorectomy patient tocompensate for the sudden loss in estrogen that occurs with ovaryremoval, and gradually decreasing the HRT dose over time. In certainembodiments, estrogen is administered at about 0.01 mg/kg to about 0.1mg/kg, and the dosage is decreased over time.

In another aspect, the invention relates to methods for treating cancerin a KRAS-variant subject, which include reducing estrogen activity inthe KRAS-variant subject to reduce the risk of aggressive tumor growth.In certain embodiments, estrogen exposure is gradually decreased byantagonizing estrogen function, for example, by administering anestrogen antagonist or an estrogen receptor antagonist. In certainembodiments, the estrogen receptor antagonist is tamoxifen. Thetamoxifen may be administered at 10 or 20 mg twice a day or at 10 or 20mg once daily. Administration may continue for three months, six months,one year, two years, three years, four years, five years, orindefinitely. In certain embodiments, the estrogen receptor antagonistis a selective estrogen receptor modulator (SERM) or selective estrogenreceptor down-regulator (SERD). In certain embodiments, the SERM isclomifene, femarelle, ormeloxifene, raloxifene, toremifene,lasofoxifene, ospemifene, afimoxifene, arzoxifene or bazedoxifene. Incertain embodiments, the SERD is fulvestrant (Faslodex®). Any dosage ofthe estrogen antagonist or estrogen receptor antagonist known in the artmay be used. Administration of the estrogen antagonist or estrogenreceptor antagonist may continue for three months, six months, one year,two years, three years, four years, five years, or indefinitely.

In any of the above embodiments, the cancer may be breast cancer,uterine cancer or ovarian cancer. In any of the above embodiments, themethod further comprises detecting a single nucleotide polymorphism(SNP) at position 4 of the let-7 complementary site 6 of KRAS in apatient sample wherein the presence of said SNP indicates an increasedrisk of developing cancer in said subject.

In another aspect, the invention relates to a method of predicting anincreased risk of developing a second, independent breast cancer in asubject. The method includes detecting a single nucleotide polymorphism(SNP) at position 4 of the let-7 complementary site 6 of KRAS in apatient sample wherein the presence of said SNP indicates an increasedrisk of developing a second, independent cancer in said subject. Incertain embodiments, the second, independent cancer is breast cancer. Incertain embodiments, the subject is a breast cancer patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows isogenic non-variant and KRAS-variant MCF10a cell linesgrown on epidermal growth factor (EGF). Non-variant cells showedrounded/cuboidal epithelial morphology, while KRAS-variant linesexhibited a mesenchymal spindle phenotype, suggesting a baselineepithelial to mesenchymal transition in the presence of theKRAS-variant.

FIG. 1B shows a panel of epithelial and mesenchymal markers measured bymRNA and Western Analysis. Levels of epithelial and mesenchymal markerswere consistent with KRAS-variant lines having undergone an epithelialto mesenchymal transition (EMT) and being more mesenchymal, withsignificantly lower E-Cadherin and Occludin, and significantly higherFibronectin and Vimentin than the non-variant line.

FIG. 1C shows that KRAS-variant cell lines (MUT1 and MUT2) were unableto form normal acini in 3D culture, unlike the non-variant (PAR) line orcontrol MCF7 line.

FIG. 1D shows that miRNA expression was significantly different betweenKRAS-variant cells (MUT) and the non-variant (PAR) cells. As shown,miR-200c was the most dramatically down-regulated miR in the MUT cells.

FIG. 1E shows that in KRAS-variant cell lines, removal of estrogen orestrogen activity leads to increased colony formation, which indicatestransformation from a non-cancer to a cancer. KRAS-variant cell linesexhibit a 2-fold increased colony formation rate in the presence ofTamoxifen (TAM), a 6.2 fold increased colony formation rate in charcoalstripped media (media from which estrogen has been removed), and a 7.9fold increased colony formation rate with both TAM and charcoal strippedmedia.

FIG. 2 shows a bar graph and Western Blot depicting that KRAS mRNA waslower in the MT cells, but KRAS protein was slightly elevated.

FIG. 3 shows representative images of MCF10A cells lines with(MCF10a^(KRAS+/−); MT1 and MT2) versus without (MCF10a^(KRAS−/−); WT)the KRAS-variant grown in soft agar. All the cell lines were seeded insoft agar of 6-tissue culture plates (7.5×10⁴ cells per well). The cellswere grown for 21 days to allow for anchorage-independent colonyformation. MCF10a WT (i) and isogenic KRAS-variant cell lines, MT1 (ii)and MT2 (iii) did not show anchorage-independent cell growth in softagar. The MCF7 (positive control; iv) formed numerous colonies. Themedia only (negative control; v) did not show any colony formation.

FIG. 4 shows that in KRAS-variant cell lines, returning estrogen tocharcoal-stripped media resulted in decreased transformation (i.e.,decreased colony formation), supporting that the increase intransformation in charcoal-stripped media was due to estrogen removal.

FIG. 5 shows that different anti-estrogen treatments lead to differenttransformation rates in anchorage independent growth assays in theKRAS-variant MCF10A lines (L1 and L2) compared to the parental (WT)MCF10A line. Estrogen treatment prevents transformation.

FIG. 6 shows a Kaplan-Meier curve showing that women with a KRAS GGmutation tend to be at higher risk of developing second primary tumors(HR=13.49, 95% C.I.=4.20-43.06, p.val<0.001). These data are derivedfrom 7 patients.

DETAILED DESCRIPTION A. Introduction

The KRAS-variant, a SNP in the 3′ untranslated region (UTR) of KRAS,referred to herein as the “LCS6 SNP,” or the “KRAS-variant,” is agerm-line, dynamically regulated microRNA binding site mutation in theKRAS oncogene, which predicts increased cancer risk primarily in women,multiple cancer risk in the same patient, and tumor biology acrosscancer types. The KRAS-variant has been previously shown to be a geneticmarker of increased risk of triple negative breast cancer and to predictaggressive breast tumor biology with hormone replacement therapy use.

The invention is based upon the unexpected discovery that for women withthe KRAS-variant, estrogen withdrawal appears to increase risk for bothtumor development as well as aggressive breast cancer tumor biology,both in vivo and in vitro. Further, the invention relates to theunexpected discovery that KRAS-variant breast cancer patients are at asignificantly elevated risk for both synchronous and asynchronous secondbreast cancer development, which is not otherwise explained by otherknown risk factors.

There are three human RAS genes comprising HRAS, KRAS, and NRAS. Eachgene comprises multiple miRNA complementary sites in the 3′UTR of theirmRNA transcripts. Specifically, each human RAS gene comprises multiplelet-7 complementary sites (LCSs). The let-7 family-of-microRNAs (miRNAs)are global genetic regulators important in controlling lung canceroncogene expression by binding to the 3′UTRs (untranslated regions) oftheir target messenger RNAs (mRNAs).

Specifically, the term “let-7 complementary site” is meant to describeany region of a gene or gene transcript that binds a member of the let-7family of miRNAs. Moreover, this term encompasses those sequences withina gene or gene transcript that are complementary to the sequence of alet-7 family miRNA. The term “complementary” describes a threshold ofbinding between two sequences wherein a majority of nucleotides in eachsequence are capable of binding to a majority of nucleotides within theother sequence in trans.

The Human KRAS 3′ UTR comprises 8 LCSs named LCS1-LCS8, respectively.For the following sequences, thymine (T) may be substituted for uracil(U). LCS1 comprises the sequence GACAGUGGAAGUUUUUUUUUCCUCG (SEQ ID NO:1). LCS2 comprises the sequence AUUAGUGUCAUCUUGCCUC (SEQ ID NO: 2). LCS3comprises the sequence AAUGCCCUACAUCUUAUUUUCCUCA (SEQ ID NO: 3). LCS4comprises the sequence GGUUCAAGCGAUUCUCGUGCCUCG (SEQ ID NO: 4). LCS5comprises the sequence GGCUGGUCCGAACUCCUGACCUCA (SEQ ID NO: 5). LCS6comprises the sequence GAUUCACCCACCUUGGCCUCA (SEQ ID NO: 6). LCS7comprises the sequence GGGUGUUAAGACUUGACACAGUACCUCG (SEQ ID NO: 7). LCS8comprises the sequence AGUGCUUAUGAGGGGAUAUUUAGGCCUC (SEQ ID NO: 8).

Human KRAS has two wild type forms, encoded by transcripts a and b,which provided below as SEQ ID NOs: 9 and 10, respectively. Thesequences of each human KRAS transcript, containing the LCS6 SNP(KRAS-variant), are provided below as SEQ ID NOs: 11 and 12.

Human KRAS, transcript variant a, is encoded by the following mRNAsequence (NCBI Accession No. NM_033360 and SEQ ID NO: 9) (untranslatedregions are bolded, LCS6 is underlined):

1 ggccgcggcg gcggaggcag cagcggcggc ggcagtggcg gcggcgaagg tggcggcggc 61tcggccagta ctcccggccc ccgccatttc ggactgggag cgagcgcggc gcaggcactg 121aaggcggcgg cggggccaga ggctcagcgg ctcccaggtg cgggagagag gcctgctgaa 181aatgactgaa tataaacttg tggtagttgg agctggtggc gtaggcaaga gtgccttgac 241gatacagcta attcagaatc attttgtgga cgaatatgat ccaacaatag aggattccta 301caggaagcaa gtagtaattg atggagaaac ctgtctcttg gatattctcg acacagcagg 361tcaagaggag tacagtgcaa tgagggacca gtacatgagg actggggagg gctttctttg 421tgtatttgcc ataaataata ctaaatcatt tgaagatatt caccattata gagaacaaat 481taaaagagtt aaggactctg aagatgtacc tatggtccta gtaggaaata aatgtgattt 541gccttctaga acagtagaca caaaacaggc tcaggactta gcaagaagtt atggaattcc 601ttttattgaa auatuagcaa agauaagaua gagagtggag gatguttttt atauattggt 661gagggagatc cgacaataca gattgaaaaa aatcagcaaa gaagaaaaga ctcctggctg 721tgtgaaaatt aaaaaatgca ttataatgta atctgggtgt tgatgatgcc ttctatacat 781tagttcgaga aattcgaaaa cataaagaaa agatgagcaa agatggtaaa aagaagaaaa 841agaagtcaaa gacaaagtgt gtaattatgt aaatacaatt tgtacttttt tcttaaggca 901tactagtaca agtggtaatt tttgtacatt acactaaatt attagcattt gttttagcat 961tacctaattt ttttcctgct ccatgcagac tgttagcttt taccttaaat gcttatttta 1021aaatgacagt ggaagttttt ttttcctcta agtgccagta ttcccagagt tttggttttt 1081gaactagcaa tgcctgtgaa aaagaaactg aatacctaag atttctgtct tggggttttt 1141ggtgcatgca gttgattact tcttattttt cttaccaatt gtgaatgttg gtgtgaaaca 1201aattaatgaa gcttttgaat catccctatt ctgtgtttta tctagtcaca taaatggatt 1261aattactaat ttcagttgag accttctaat tggtttttac tgaaacattg agggaacaca 1321aatttatggg cttcctgatg atgattcttc taggcatcat gtcctatagt ttgtcatccc 1381tgatgaatgt aaagttacac tgttcacaaa ggttttgtct cctttccact gctattagtc 1441atggtcactc tccccaaaat attatatttt ttctataaaa agaaaaaaat ggaaaaaaat 1501tacaaggcaa tggaaactat tataaggcca tttccttttc acattagata aattactata 1561aagactccta atagcttttc ctgttaaggc agacccagta tgaaatgggg attattatag 1621caaccatttt ggggctatat ttacatgcta ctaaattttt ataataattg aaaagatttt 1681aacaagtata aaaaattctc ataggaatta aatgtagtct ccctgtgtca gactgctctt 1741tcatagtata actttaaatc ttttcttcaa cttgagtctt tgaagatagt tttaattctg 1801cttgtgacat taaaagatta tttgggccag ttatagctta ttaggtgttg aagagaccaa 1861ggttgcaagg ccaggccctg tgtgaacctt tgagctttca tagagagttt cacagcatgg 1921actgtgtccc cacggtcatc cagtgttgtc atgcattggt tagtcaaaat ggggagggac 1981tagggcagtt tggatagctc aacaagatac aatctcactc tgtggtggtc ctgctgacaa 2041atcaagagca ttgcttttgt ttcttaagaa aacaaactct tttttaaaaa ttacttttaa 2101atattaactc aaaagttgag attttggggt ggtggtgtgc caagacatta attttttttt 2161taaacaatga agtgaaaaag ttttacaatc tctaggtttg gctagttctc ttaacactgg 2221ttaaattaac attgcataaa cacttttcaa gtctgatcca tatttaataa tgctttaaaa 2281taaaaataaa aacaatcctt ttgataaatt taaaatgtta cttattttaa aataaatgaa 2341gtgagatggc atggtgaggt gaaagtatca ctggactagg aagaaggtga cttaggttct 2401agataggtgt cttttaggac tctgattttg aggacatcac ttactatcca tttcttcatg 2461ttaaaagaag tcatctcaaa ctcttagttt ttttttttta caactatgta atttatattc 2521catttacata aggatacact tatttgtcaa gctcagcaca atctgtaaat ttttaaccta 2581tgttacacca tcttcagtgc cagtcttggg caaaattgtg caagaggtga agtttatatt 2641tgaatatcca ttctcgtttt aggactcttc ttccatatta gtgtcatctt gcctccctac 2701cttccacatg ccccatgact tgatgcagtt ttaatacttg taattcccct aaccataaga 2761tttactgctg ctgtggatat ctccatgaag ttttcccact gagtcacatc agaaatgccc 2821tacatcttat ttcctcaggg ctcaagagaa tctgacagat accataaagg gatttgacct 2881aatcactaat tttcaggtgg tggctgatgc tttgaacatc tctttgctgc ccaatccatt 2941agcgacagta ggatttttca aacctggtat gaatagacag aaccctatcc agtggaagga 3001gaatttaata aagatagtgc tgaaagaatt ccttaggtaa tctataacta ggactactcc 3061tggtaacagt aatacattcc attgttttag taaccagaaa tcttcatgca atgaaaaata 3121ctttaattca tgaagcttac tttttttttt tggtgtcaga gtctcgctct tgtcacccag 3181gctggaatgc agtggcgcca tctcagctca ctgcaacctc catctcccag gttcaagcga 3241ttctcgtgcc tcggcctcct gagtagctgg gattacaggc gtgtgccact acactcaact 3301aatttttgta tttttaggag agacggggtt tcaccctgtt ggccaggctg gtctcgaact 3361cctgacctca  agtgattcac ccaccttggc ctca taaacc tgttttgcag aactcattta 3421ttcagcaaat atttattgag tgcctaccag atgccagtca ccgcacaagg cactgggtat 3481atggtatccc caaacaagag acataatccc ggtccttagg tagtgctagt gtggtctgta 3541atatcttact aaggcctttg gtatacgacc cagagataac acgatgcgta ttttagtttt 3601gcaaagaagg ggtttggtct ctgtgccagc tctataattg ttttgctacg attccactga 3661aactcttcga tcaagctact ttatgtaaat cacttcattg ttttaaagga ataaacttga 3721ttatattgtt tttttatttg gcataactgt gattctttta ggacaattac tgtacacatt 3781aaggtgtatg tcagatattc atattgaccc aaatgtgtaa tattccagtt ttctctgcat 3841aagtaattaa aatatactta aaaattaata gttttatctg ggtacaaata aacaggtgcc 3901tgaactagtt cacagacaag gaaacttcta tgtaaaaatc actatgattt ctgaattgct 3961atgtgaaact acagatcttt ggaacactgt ttaggtaggg tgttaagact tacacagtac 4021ctcgtttcta cacagagaaa gaaatggcca tacttcagga actgcagtgc ttatgagggg 4081atatttaggc ctcttgaatt tttgatgtag atgggcattt ttttaaggta gtggttaatt 4141acctttatgt gaactttgaa tggtttaaca aaagatttgt ttttgtagag attttaaagg 4201gggagaattc tagaaataaa tgttacctaa ttattacagc cttaaagaca aaaatccttg 4261ttgaagtttt tttaaaaaaa gctaaattac atagacttag gcattaacat gtttgtggaa 4321gaatatagca gacgtatatt gtatcatttg agtgaatgtt cccaagtagg cattctaggc 4381tctatttaac tgagtcacac tgcataggaa tttagaacct aacttttata ggttatcaaa 4441actgttgtca ccattgcaca attttgtcct aatatataca tagaaacttt gtggggcatg 4501ttaagttaca gtttgcacaa gttcatctca tttgtattcc attgattttt tttttcttct 4561aaacattttt tcttcaaaca gtatataact ttttttaggg gatttttttt tagacagcaa 4621aaactatctg aagatttcca tttgtcaaaa agtaatgatt tcttgataat tgtgtagtaa 4681tgttttttag aacccagcag ttaccttaaa gctgaattta tatttagtaa cttctgtgtt 4741aatactggat agcatgaatt ctgcattgag aaactgaata gctgtcataa aatgaaactt 4801tctttctaaa gaaagatact cacatgagtt cttgaagaat agtcataact agattaagat 4861ctgtgtttta gtttaatagt ttgaagtgcc tgtttgggat aatgataggt aatttagatg 4921aatttagggg aaaaaaaagt tatctgcaga tatgttgagg gcccatctct ccccccacac 4981ccccacagag ctaactgggt tacagtgttt tatccgaaag tttccaattc cactgtcttg 5041tgttttcatg ttgaaaatac ttttgcattt ttcctttgag tgccaatttc ttactagtac 5101tatttcttaa tgtaacatgt ttacctggaa tgtattttaa ctatttttgt atagtgtaaa 5161ctgaaacatg cacattttgt acattgtgct ttcttttgtg ggacatatgc agtgtgatcc 5221agttgttttc catcatttgg ttgcgctgac ctaggaatgt tggtcatatc aaacattaaa 5281aatgaccact cttttaattg aaattaactt ttaaatgttt ataggagtat gtgctgtgaa 5341gtgatctaaa atttgtaata tttttgtcat gaactgtact actcctaatt attgtaatgt 5401aataaaaata gttacagtga caaaaaaaaa aaaaaa

Human ERAS, transcript variant b, is encoded by the following mRNAsequence (NCBI Accession No. NM_004985 and SEQ ID NO: 10) (untranslatedregions are bolded, LCS6 is underlined):

1 ggccgcggcg gcggaggcag cagcggcggc ggcagtggcg gcggcgaagg tggcggcggc 61tcggccagta ctcccggccc ccgccatttc ggactgggag cgagcgcggc gcaggcactg 121aaggcggcgg cggggccaga ggctcagcgg ctcccaggtg cgggagagag gcctgctgaa 181aatgactgaa tataaacttg tggtagttgg agctggtggc gtaggcaaga gtgccttgac 241gatacagcta attcagaatc attttgtgga cgaatatgat ccaacaatag aggattccta 301caggaagcaa gtagtaattg atggagaaac ctgtctcttg gatattctcg acacagcagg 361tcaagaggag tacagtgcaa tgagggacca gtacatgagg actggggagg gctttctttg 421tgtatttgcc ataaataata ctaaatcatt tgaagatatt caccattata gagaacaaat 481taaaagagtt aaggactctg aagatgtacc tatggtccta gtaggaaata aatgtgattt 541gccttctaga acagtagaca caaaacaggc tcaggactta gcaagaagtt atggaattcc 601ttttattgaa acatcagcaa agacaagaca gggtgttgat gatgccttct atacattagt 661tcgagaaatt cgaaaacata aagaaaagat gagcaaagat ggtaaaaaga agaaaaagaa 721gtcaaagaca aagtgtgtaa ttatgtaaat acaatttgta cttttttctt aaggcatact 781agtacaagtg gtaatttttg tacattacac taaattatta gcatttgttt tagcattacc 841taattttttt cctgctccat gcagactgtt agcttttacc ttaaatgctt attttaaaat 901gacagtggaa gttttttttt cctctaagtg ccagtattcc cagagttttg gtttttgaac 961tagcaatgcc tgtgaaaaag aaactgaata cctaagattt ctgtcttggg gtttttggtg 1021catgcagttg attacttctt atttttctta ccaattgtga atgttggtgt gaaacaaatt 1081aatgaagctt ttgaatcatc cctattctgt gttttatcta gtcacataaa tggattaatt 1141actaatttca gttgagacct tctaattggt ttttactgaa acattgaggg aacacaaatt 1201tatgggcttc ctgatgatga ttcttctagg catcatgtcc tatagtttgt catccctgat 1261gaatgtaaag ttacactgtt cacaaaggtt ttgtctcctt tccactgcta ttagtcatgg 1321tcactctccc caaaatatta tattttttct ataaaaagaa aaaaatggaa aaaaattaca 1381aggcaatgga aactattata aggccatttc cttttcacat tagataaatt actataaaga 1441ctcctaatag cttttcctgt taaggcagac ccagtatgaa atggggatta ttatagcaac 1501cattttgggg ctatatttac atgctactaa atttttataa taattgaaaa gattttaaca 1561agtataaaaa attctcatag gaattaaatg tagtctccct gtgtcagact gctctttcat 1621agtataactt taaatctttt cttcaacttg agtctttgaa gatagtttta attctgcttg 1681tgacattaaa agattatttg ggccagttat agcttattag gtgttgaaga gaccaaggtt 1741gcaaggccag gccctgtgtg aacctttgag ctttcataga gagtttcaca gcatggactg 1801tgtccccacg gtcatccagt gttgtcatgc attggttagt caaaatgggg agggactagg 1861gcagtttgga tagctcaaca agatacaatc tcactctgtg gtggtcctgc tgacaaatca 1921agagcattgc ttttgtttct taagaaaaca aactcttttt taaaaattac ttttaaatat 1981taactcaaaa gttgagattt tggggtggtg gtgtgccaag acattaattt tttttttaaa 2041caatgaagtg aaaaagtttt acaatctcta ggtttggcta gttctcttaa cactggttaa 2101attaacattg cataaacact tttcaagtct gatccatatt taataatgct ttaaaataaa 2161aataaaaaca atccttttga taaatttaaa atgttactta ttttaaaata aatgaagtga 2221gatggcatgg tgaggtgaaa gtatcactgg actaggaaga aggtgactta ggttctagat 2281aggtgtcttt taggactctg attttgagga catcacttac tatccatttc ttcatgttaa 2341aagaagtcat ctcaaactct tagttttttt tttttacaac tatgtaattt atattccatt 2401tacataagga tacacttatt tgtcaagctc agcacaatct gtaaattttt aacctatgtt 2461acaccatctt cagtgccagt cttgggcaaa attgtgcaag aggtgaagtt tatatttgaa 2521tatccattct cgttttagga ctcttcttcc atattagtgt catcttgcct ccctaccttc 2581cacatgcccc atgacttgat gcagttttaa tacttgtaat tcccctaacc ataagattta 2641ctgctgctgt ggatatctcc atgaagtttt cccactgagt cacatcagaa atgccctaca 2701tcttatttcc tcagggctca agagaatctg acagatacca taaagggatt tgacctaatc 2761actaattttc aggtggtggc tgatgctttg aacatctctt tgctgcccaa tccattagcg 2821acagtaggat ttttcaaacc tggtatgaat agacagaacc ctatccagtg gaaggagaat 2881ttaataaaga tagtgctgaa agaattcctt aggtaatcta taactaggac tactcctggt 2941aacagtaata cattccattg ttttagtaac cagaaatctt catgcaatga aaaatacttt 3001aattcatgaa gcttactttt tttttttggt gtcagagtct cgctcttgtc acccaggctg 3061gaatgcagtg gcgccatctc agctcactgc aacctccatc tcccaggttc aagcgattct 3121cgtgcctcgg cctcctgagt agctgggatt acaggcgtgt gccactacac tcaactaatt 3181tttgtatttt taggagagac ggggtttcac cctgttggcc aggctggtct cgaactcctg 3241acctcaagt g attcacccac cttggcctca  taaacctgtt ttgcagaact catttattca 3301gcaaatattt attgagtgcc taccagatgc cagtcaccgc acaaggcact gggtatatgg 3361tatccccaaa caagagacat aatcccggtc cttaggtagt gctagtgtgg tctgtaatat 3421cttactaagg cctttggtat acgacccaga gataacacga tgcgtatttt agttttgcaa 3481agaaggggtt tggtctctgt gccagctcta taattgtttt gctacgattc cactgaaact 3541cttcgatcaa gctactttat gtaaatcact tcattgtttt aaaggaataa acttgattat 3601attgtttttt tatttggcat aactgtgatt cttttaggac aattactgta cacattaagg 3661tgtatgtcag atattcatat tgacccaaat gtgtaatatt ccagttttct ctgcataagt 3721aattaaaata tacttaaaaa ttaatagttt tatctgggta caaataaaca ggtgcctgaa 3781ctagttcaca gacaaggaaa cttctatgta aaaatcacta tgatttctga attgctatgt 3841gaaactacag atctttggaa cactgtttag gtagggtgtt aagacttaca cagtacctcg 3901tttctacaca gagaaagaaa tggccatact tcaggaactg cagtgcttat gaggggatat 3961ttaggcctct tgaatttttg atgtagatgg gcattttttt aaggtagtgg ttaattacct 4021ttatgtgaac tttgaatggt ttaacaaaag atttgttttt gtagagattt taaaggggga 4081gaattctaga aataaatgtt acctaattat tacagcctta aagacaaaaa tccttgttga 4141agttttttta aaaaaagcta aattacatag acttaggcat taacatgttt gtggaagaat 4201atagcagacg tatattgtat catttgagtg aatgttccca agtaggcatt ctaggctcta 4261tttaactgag tcacactgca taggaattta gaacctaact tttataggtt atcaaaactg 4321ttgtcaccat tgcacaattt tgtcctaata tatacataga aactttgtgg ggcatgttaa 4381gttacagttt gcacaagttc atctcatttg tattccattg attttttttt tcttctaaac 4441attttttctt caaacagtat ataacttttt ttaggggatt tttttttaga cagcaaaaac 4501tatctgaaga tttccatttg tcaaaaagta atgatttctt gataattgtg tagtaatgtt 4561ttttagaacc cagcagttac cttaaagctg aatttatatt tagtaacttc tgtgttaata 4621ctggatagca tgaattctgc attgagaaac tgaatagctg tcataaaatg aaactttctt 4681tctaaagaaa gatactcaca tgagttcttg aagaatagtc ataactagat taagatctgt 4741gttttagttt aatagtttga agtgcctgtt tgggataatg ataggtaatt tagatgaatt 4801taggggaaaa aaaagttatc tgcagatatg ttgagggccc atctctcccc ccacaccccc 4861acagagctaa ctgggttaca gtgttttatc cgaaagtttc caattccact gtcttgtgtt 4921ttcatgttga aaatactttt gcatttttcc tttgagtgcc aatttcttac tagtactatt 4981tcttaatgta acatgtttac ctggaatgta ttttaactat ttttgtatag tgtaaactga 5041aacatgcaca ttttgtacat tgtgctttct tttgtgggac atatgcagtg tgatccagtt 5101gttttccatc atttggttgc gctgacctag gaatgttggt catatcaaac attaaaaatg 5161accactcttt taattgaaat taacttttaa atgtttatag gagtatgtgc tgtgaagtga 5221tctaaaattt gtaatatttt tgtcatgaac tgtactactc ctaattattg taatgtaata 5281aaaatagtta cagtgacaaa aaaaaaaaaa aa

Human KRAS, transcript variant a, comprising the LCS6 SNP(KRAS-variant), is encoded by the following mRNA sequence (SEQ ID NO:11) (untranslated regions are bolded, LCS6 is underlined, SNP iscapitalized):

1 ggccgcggcg gcggaggcag cagcggcggc ggcagtggcg gcggcgaagg tggcggcggc 61tcggccagta ctcccggccc ccgccatttc ggactgggag cgagcgcggc gcaggcactg 121aaggcggcgg cggggccaga ggctcagcgg ctcccaggtg cgggagagag gcctgctgaa 181aatgactgaa tataaacttg tggtagttgg agctggtggc gtaggcaaga gtgccttgac 241gatacagcta attcagaatc attttgtgga cgaatatgat ccaacaatag aggattccta 301caggaaguaa gtagtaattg atggagaaac utgtctuttg gatattutug acacaguagg 361tcaagaggag tacagtgcaa tgagggacca gtacatgagg actggggagg gctttctttg 421tgtatttgcc ataaataata ctaaatcatt tgaagatatt caccattata gagaacaaat 481taaaagagtt aaggactctg aagatgtacc tatggtccta gtaggaaata aatgtgattt 541gccttctaga acagtagaca caaaacaggc tcaggactta gcaagaagtt atggaattcc 601ttttattgaa acatcagcaa agacaagaca gagagtggag gatgcttttt atacattggt 661gagggagatc cgacaataca gattgaaaaa aatcagcaaa gaagaaaaga ctcctggctg 721tgtgaaaatt aaaaaatgca ttataatgta atctgggtgt tgatgatgcc ttctatacat 781tagttcgaga aattcgaaaa cataaagaaa agatgagcaa agatggtaaa aagaagaaaa 841agaagtcaaa gacaaagtgt gtaattatgt aaatacaatt tgtacttttt tcttaaggca 901tactagtaca agtggtaatt tttgtacatt acactaaatt attagcattt gttttagcat 961tacctaattt ttttcctgct ccatgcagac tgttagcttt taccttaaat gcttatttta 1021aaatgacagt ggaagttttt ttttcctcta agtgccagta ttcccagagt tttggttttt 1081gaactagcaa tgcctgtgaa aaagaaactg aatacctaag atttctgtct tggggttttt 1141ggtgcatgca gttgattact tcttattttt cttaccaatt gtgaatgttg gtgtgaaaca 1201aattaatgaa gcttttgaat catccctatt ctgtgtttta tctagtcaca taaatggatt 1261aattactaat ttcagttgag accttctaat tggtttttac tgaaacattg agggaacaca 1321aatttatggg cttcctgatg atgattcttc taggcatcat gtcctatagt ttgtcatccc 1381tgatgaatgt aaagttacac tgttcacaaa ggttttgtct cctttccact gctattagtc 1441atggtcactc tccccaaaat attatatttt ttctataaaa agaaaaaaat ggaaaaaaat 1501tacaaggcaa tggaaactat tataaggcca tttccttttc acattagata aattactata 1561aagactccta atagcttttc ctgttaaggc agacccagta tgaaatgggg attattatag 1621caaccatttt ggggctatat ttacatgcta ctaaattttt ataataattg aaaagatttt 1681aacaagtata aaaaattctc ataggaatta aatgtagtct ccctgtgtca gactgctctt 1741tcatagtata actttaaatc ttttcttcaa cttgagtctt tgaagatagt tttaattctg 1801cttgtgacat taaaagatta tttgggccag ttatagctta ttaggtgttg aagagaccaa 1861ggttgcaagg ccaggccctg tgtgaacctt tgagctttca tagagagttt cacagcatgg 1921actgtgtccc cacggtcatc cagtgttgtc atgcattggt tagtcaaaat ggggagggac 1981tagggcagtt tggatagctc aacaagatac aatctcactc tgtggtggtc ctgctgacaa 2041atcaagagca ttgcttttgt ttcttaagaa aacaaactct tttttaaaaa ttacttttaa 2101atattaactc aaaagttgag attttggggt ggtggtgtgc caagacatta attttttttt 2161taaacaatga agtgaaaaag ttttacaatc tctaggtttg gctagttctc ttaacactgg 2221ttaaattaac attgcataaa cacttttcaa gtctgatcca tatttaataa tgctttaaaa 2281taaaaataaa aacaatcctt ttgataaatt taaaatgtta cttattttaa aataaatgaa 2341gtgagatggc atggtgaggt gaaagtatca ctggactagg aagaaggtga cttaggttct 2401agataggtgt cttttaggac tctgattttg aggacatcac ttactatcca tttcttcatg 2461ttaaaagaag tcatctcaaa ctcttagttt ttttttttta caactatgta atttatattc 2521catttacata aggatacact tatttgtcaa gctcagcaca atctgtaaat ttttaaccta 2581tgttacacca tcttcagtgc cagtcttggg caaaattgtg caagaggtga agtttatatt 2641tgaatatcca ttctcgtttt aggactcttc ttccatatta gtgtcatctt gcctccctac 2701cttccacatg ccccatgact tgatgcagtt ttaatacttg taattcccct aaccataaga 2761tttactgctg ctgtggatat ctccatgaag ttttcccact gagtcacatc agaaatgccc 2821tacatcttat ttcctcaggg ctcaagagaa tctgacagat accataaagg gatttgacct 2881aatcactaat tttcaggtgg tggctgatgc tttgaacatc tctttgctgc ccaatccatt 2941agcgacagta ggatttttca aacctggtat gaatagacag aaccctatcc agtggaagga 3001gaatttaata aagatagtgc tgaaagaatt ccttaggtaa tctataacta ggactactcc 3061tggtaacagt aatacattcc attgttttag taaccagaaa tcttcatgca atgaaaaata 3121ctttaattca tgaagcttac tttttttttt tggtgtcaga gtctcgctct tgtcacccag 3181gctggaatgc agtggcgcca tctcagctca ctgcaacctc catctcccag gttcaagcga 3241ttctcgtgcc tcggcctcct gagtagctgg gattacaggc gtgtgccact acactcaact 3301aatttttgta tttttaggag agacggggtt tcaccctgtt ggccaggctg gtctcgaact 3361cctgacctca agt gatGcac ccaccttggc ctca taaacc tgttttgcag aactcattta 3421ttcagcaaat atttattgag tgcctaccag atgccagtca ccgcacaagg cactgggtat 3481atggtatccc caaacaagag acataatccc ggtccttagg tagtgctagt gtggtctgta 3541atatcttact aaggcctttg gtatacgacc cagagataac acgatgcgta ttttagtttt 3601gcaaagaagg ggtttggtct ctgtgccagc tctataattg ttttgctacg attccactga 3661aactcttcga tcaagctact ttatgtaaat cacttcattg ttttaaagga ataaacttga 3721ttatattgtt tttttatttg gcataactgt gattctttta ggacaattac tgtacacatt 3781aaggtgtatg tcagatattc atattgaccc aaatgtgtaa tattccagtt ttctctgcat 3841aagtaattaa aatatactta aaaattaata gttttatctg ggtacaaata aacaggtgcc 3901tgaactagtt cacagacaag gaaacttcta tgtaaaaatc actatgattt ctgaattgct 3961atgtgaaact acagatcttt ggaacactgt ttaggtaggg tgttaagact tacacagtac 4021ctcgtttcta cacagagaaa gaaatggcca tacttcagga actgcagtgc ttatgagggg 4081atatttaggc ctcttgaatt tttgatgtag atgggcattt ttttaaggta gtggttaatt 4141acctttatgt gaactttgaa tggtttaaca aaagatttgt ttttgtagag attttaaagg 4201gggagaattc tagaaataaa tgttacctaa ttattacagc cttaaagaca aaaatccttg 4261ttgaagtttt tttaaaaaaa gctaaattac atagacttag gcattaacat gtttgtggaa 4321gaatatagca gacgtatatt gtatcatttg agtgaatgtt cccaagtagg cattctaggc 4381tctatttaac tgagtcacac tgcataggaa tttagaacct aacttttata ggttatcaaa 4441actgttgtca ccattgcaca attttgtcct aatatataca tagaaacttt gtggggcatg 4501ttaagttaca gtttgcacaa gttcatctca tttgtattcc attgattttt tttttcttct 4561aaacattttt tcttcaaaca gtatataact ttttttaggg gatttttttt tagacagcaa 4621aaactatctg aagatttcca tttgtcaaaa agtaatgatt tcttgataat tgtgtagtaa 4681tgttttttag aacccagcag ttaccttaaa gctgaattta tatttagtaa cttctgtgtt 4741aatactggat agcatgaatt ctgcattgag aaactgaata gctgtcataa aatgaaactt 4801tctttctaaa gaaagatact cacatgagtt cttgaagaat agtcataact agattaagat 4861ctgtgtttta gtttaatagt ttgaagtgcc tgtttgggat aatgataggt aatttagatg 4921aatttagggg aaaaaaaagt tatctgcaga tatgttgagg gcccatctct ccccccacac 4981ccccacagag ctaactgggt tacagtgttt tatccgaaag tttccaattc cactgtcttg 5041tgttttcatg ttgaaaatac ttttgcattt ttcctttgag tgccaatttc ttactagtac 5101tatttcttaa tgtaacatgt ttacctggaa tgtattttaa ctatttttgt atagtgtaaa 5161ctgaaacatg cacattttgt acattgtgct ttcttttgtg ggacatatgc agtgtgatcc 5221agttgttttc catcatttgg ttgcgctgac ctaggaatgt tggtcatatc aaacattaaa 5281aatgaccact cttttaattg aaattaactt ttaaatgttt ataggagtat gtgctgtgaa 5341gtgatctaaa atttgtaata tttttgtcat gaactgtact actcctaatt attgtaatgt 5401aataaaaata gttacagtga caaaaaaaaa aaaaaa

Human KRAS, transcript variant b, comprising the LCS6 SNP(KRAS-variant), is encoded by the following mRNA sequence (SEQ TD NO:12) (untranslated regions are bolded, LCS6 is underlined, SNP iscapitalized):

1 ggccgcggcg gcggaggcag cagcggcggc ggcagtggcg gcggcgaagg tggcggcggc 61tcggccagta ctcccggccc ccgccatttc ggactgggag cgagcgcggc gcaggcactg 121aaggcggcgg cggggccaga ggctcagcgg ctoccaggtg cgggagagag gcctgctgaa 181aatgactgaa tataaacttg tggtagttgg agctggtggc gtaggcaaga gtgccttgac 241gatacagcta attcagaatc attttgtgga cgaatatgat ccaacaatag aggattccta 301caggaagcaa gtagtaattg atggagaaac ctgtctcttg gatattctcg acacagcagg 361tcaagaggag tacagtgcaa tgagggacca gtacatgagg actggggagg gctttctttg 421tgtatttgcc ataaataata ctaaatcatt tgaagatatt caccattata gagaacaaat 481taaaagagtt aaggactctg aagatgtacc tatggtccta gtaggaaata aatgtgattt 541gccttctaga acagtagaca caaaacaggc tcaggactta gcaagaagtt atggaattcc 601ttttattgaa acatcagcaa agacaagaca gggtgttgat gatgccttct atacattagt 661tcgagaaatt cgaaaacata aagaaaagat gagcaaagat ggtaaaaaga agaaaaagaa 721gtcaaagaca aagtgtgtaa ttatgtaaat acaatttgta cttttttctt aaggcatact 781agtacaagtg gtaatttttg tacattacac taaattatta gcatttgttt tagcattacc 841taattttttt cctgctccat gcagactgtt agcttttacc ttaaatgctt attttaaaat 901gacagtggaa gttttttttt cctctaagtg ccagtattcc cagagttttg gtttttgaac 961tagcaatgcc tgtgaaaaag aaactgaata cctaagattt ctgtcttggg gtttttggtg 1021catgcagttg attacttctt atttttctta ccaattgtga atgttggtgt gaaacaaatt 1081aatgaagctt ttgaatcatc cctattctgt gttttatcta gtcacataaa tggattaatt 1141actaatttca gttgagacct tctaattggt ttttactgaa acattgaggg aacacaaatt 1201tatgggcttc ctgatgatga ttcttctagg catcatgtcc tatagtttgt catccctgat 1261gaatgtaaag ttacactgtt cacaaaggtt ttgtctcctt tccactgcta ttagtcatgg 1321tcactctccc caaaatatta tattttttct ataaaaagaa aaaaatggaa aaaaattaca 1381aggcaatgga aactattata aggccatttc cttttcacat tagataaatt actataaaga 1441ctcctaatag cttttcctgt taaggcagac ccagtatgaa atggggatta ttatagcaac 1501cattttgggg ctatatttac atgctactaa atttttataa taattgaaaa gattttaaca 1561agtataaaaa attctcatag gaattaaatg tagtctccct gtgtcagact gctctttcat 1621agtataactt taaatctttt cttcaacttg agtctttgaa gatagtttta attctgcttg 1681tgacattaaa agattatttg ggccagttat agcttattag gtgttgaaga gaccaaggtt 1741gcaaggccag gccctgtgtg aacctttgag ctttcataga gagtttcaca gcatggactg 1801tgtccccacg gtcatccagt gttgtcatgc attggttagt caaaatgggg agggactagg 1861gcagtttgga tagctcaaca agatacaatc tcactctgtg gtggtcctgc tgacaaatca 1921agagcattgc ttttgtttct taagaaaaca aactcttttt taaaaattac ttttaaatat 1981taactcaaaa gttgagattt tggggtggtg gtgtgccaag acattaattt tttttttaaa 2041caatgaagtg aaaaagtttt acaatctcta ggtttggcta gttctcttaa cactggttaa 2101attaacattg cataaacact tttcaagtct gatccatatt taataatgct ttaaaataaa 2161aataaaaaca atccttttga taaatttaaa atgttactta ttttaaaata aatgaagtga 2221gatggcatgg tgaggtgaaa gtatcactgg actaggaaga aggtgactta ggttctagat 2281aggtgtcttt taggactctg attttgagga catcacttac tatccatttc ttcatgttaa 2341aagaagtcat ctcaaactct tagttttttt tttttacaac tatgtaattt atattccatt 2401tacataagga tacacttatt tgtcaagctc agcacaatct gtaaattttt aacctatgtt 2461acaccatctt cagtgccagt cttgggcaaa attgtgcaag aggtgaagtt tatatttgaa 2521tatccattct cgttttagga ctcttcttcc atattagtgt catcttgcct ccctaccttc 2581cacatgcccc atgacttgat gcagttttaa tacttgtaat tcccctaacc ataagattta 2641ctgctgctgt ggatatctcc atgaagtttt cccactgagt cacatcagaa atgccctaca 2701tcttatttcc tcagggctca agagaatctg acagatacca taaagggatt tgacctaatc 2761actaattttc aggtggtggc tgatgctttg aacatctctt tgctgcccaa tccattagcg 2821acagtaggat ttttcaaacc tggtatgaat agacagaacc ctatccagtg gaaggagaat 2881ttaataaaga tagtgctgaa agaattcctt aggtaatcta taactaggac tactcctggt 2941aacagtaata cattccattg ttttagtaac cagaaatctt catgcaatga aaaatacttt 3001aattcatgaa gcttactttt tttttttggt gtcagagtct cgctcttgtc acccaggctg 3061gaatgcagtg gcgccatctc agctcactgc aacctccatc tcccaggttc aagcgattct 3121cgtgcctcgg cctcctgagt agctgggatt acaggcgtgt gccactacac tcaactaatt 3181tttgtatttt taggagagac ggggtttcac cctgttggcc aggctggtct cgaactcctg 3241acctcaagt g atGcacccac cttggcctca  taaacctgtt ttgcagaact catttattca 3301gcaaatattt attgagtgcc taccagatgc cagtcaccgc acaaggcact gggtatatgg 3361tatccccaaa caagagacat aatcccggtc cttaggtagt gctagtgtgg tctgtaatat 3421cttactaagg cctttggtat acgacccaga gataacacga tgcgtatttt agttttgcaa 3481agaaggggtt tggtctctgt gccagctcta taattgtttt gctacgattc cactgaaact 3541cttcgatcaa gctactttat gtaaatcact tcattgtttt aaaggaataa acttgattat 3601attgtttttt tatttggcat aactgtgatt cttttaggac aattactgta cacattaagg 3661tgtatgtcag atattcatat tgacccaaat gtgtaatatt ccagttttct ctgcataagt 3721aattaaaata tacttaaaaa ttaatagttt tatctgggta caaataaaca ggtgcctgaa 3781ctagttcaca gacaaggaaa cttctatgta aaaatcacta tgatttctga attgctatgt 3841gaaactacag atctttggaa cactgtttag gtagggtgtt aagacttaca cagtacctcg 3901tttctacaca gagaaagaaa tggccatact tcaggaactg cagtgcttat gaggggatat 3961ttaggcctct tgaatttttg atgtagatgg gcattttttt aaggtagtgg ttaattacct 4021ttatgtgaac tttgaatggt ttaacaaaag atttgttttt gtagagattt taaaggggga 4081gaattctaga aataaatgtt acctaattat tacagcctta aagacaaaaa tccttgttga 4141agttttttta aaaaaagcta aattacatag acttaggcat taacatgttt gtggaagaat 4201atagcagacg tatattgtat catttgagtg aatgttccca agtaggcatt ctaggctcta 4261tttaactgag tcacactgca taggaattta gaacctaact tttataggtt atcaaaactg 4321ttgtcaccat tgcacaattt tgtcctaata tatacataga aactttgtgg ggcatgttaa 4381gttacagttt gcacaagttc atctcatttg tattccattg attttttttt tcttctaaac 4441attttttctt caaacagtat ataacttttt ttaggggatt tttttttaga cagcaaaaac 4501tatctgaaga tttccatttg tcaaaaagta atgatttctt gataattgtg tagtaatgtt 4561ttttagaacc cagcagttac cttaaagctg aatttatatt tagtaacttc tgtgttaata 4621ctggatagca tgaattctgc attgagaaac tgaatagctg tcataaaatg aaactttctt 4681tctaaagaaa gatactcaca tgagttcttg aagaatagtc ataactagat taagatctgt 4741gttttagttt aatagtttga agtgcctgtt tgggataatg ataggtaatt tagatgaatt 4801taggggaaaa aaaagttatc tgcagatatg ttgagggccc atctctcccc ccacaccccc 4861acagagctaa ctgggttaca gtgttttatc cgaaagtttc caattccact gtcttgtgtt 4921ttcatgttga aaatactttt gcatttttcc tttgagtgcc aatttcttac tagtactatt 4981tcttaatgta acatgtttac ctggaatgta ttttaactat ttttgtatag tgtaaactga 5041aacatgcaca ttttgtacat tgtgctttct tttgtgggac atatgcagtg tgatccagtt 5101gttttccatc atttggttgc gctgacctag gaatgttggt catatcaaac attaaaaatg 5161accactcttt taattgaaat taacttttaa atgtttatag gagtatgtgc tgtgaagtga 5221tctaaaattt gtaatatttt tgtcatgaac tgtactactc ctaattattg taatgtaata 5281aaaatagtta cagtgacaaa aaaaaaaaaa aa

The present invention encompasses a SNP within the 3′UTR of KRAS.Specifically, this SNP is the result of a substitution of a G for a U atposition 4 of SEQ ID NO: 6 of LCS6. This LCS6 SNP (KRAS-variant)comprises the sequence GAUGCACCCACCUUGGCCUCA (SNP bolded for emphasis)(SEQ ID NO: 13).

The KRAS-variant leads to altered KRAS expression by disrupting themiRNA regulation of a KRAS. The identification and characterization ofthe KRAS-variant is further described in International Application No.PCT/US08/65302 (WO 2008/151004), the contents of which are incorporatedby reference in its entirety.

B. Methods of Prevention and Treatment of Cancer

The present inventors discovered that estrogen withdrawal in subjectswith the KRAS-variant increases risk for both tumor development as wellas aggressive breast cancer tumor biology, both in vivo and in vitro.Accordingly, the present invention relates to methods for preventingcancer in a KRAS-variant subject which include administering to theKRAS-variant subject an amount of estrogen effective to reduce the riskof developing cancer. For example, the method may include administeringto a KRAS-variant subject a tapering dose of estrogen, starting with thepatient's pre-oophorectomy baseline estrogen levels, or pre-chemotherapyestrogen levels, which can be calculated. Estrogen may be administeredas any HRT or birth control formulation known in the art. In certainembodiments, the dose of estrogen administered starts at 0.1 mg/kgestrogen, and the dosage is decreased over time.

In another aspect, the invention relates to methods for treating cancerin a KRAS-variant subject, which include reducing estrogen activity inthe KRAS-variant subject to reduce the risk of aggressive tumor growth.In certain embodiments, estrogen exposure is gradually decreased byantagonizing estrogen function, for example, by administering anestrogen antagonist or an estrogen receptor antagonist. In certainembodiments, the estrogen receptor antagonist is tamoxifen. Thetamoxifen may be administered at 10 or 20 mg twice a day for threemonths or at 10 or 20 mg once daily for three months. In certainembodiments, the estrogen receptor antagonist is a selective estrogenreceptor modulator (SERM) or selective estrogen receptor down-regulator(SERD). In certain embodiments, the SERM is clomifene, femarelle,ormeloxifene, raloxifene, toremifene, lasofoxifene, ospemifene,afimoxifene, arzoxifene or bazedoxifene. In certain embodiments, theSERD is fulvestrant (Faslodex®). Any dosage of the estrogen antagonistor estrogen receptor antagonist known in the art may be used.Administration of the estrogen antagonist or estrogen receptorantagonist may continue for three months, six months, one year, twoyears, three years, four years, five years, or indefinitely.

Gradually decreasing estrogen function can include reducing the amountof HRT or estrogen administered over a period of, e.g., one week, twoweeks, one to six weeks, one month, two months, one to six months, twoto six months, six months to a year, one year, two years, three years,four years and five years. The amount of HRT or estrogen administeredcan be reduced to zero, or to a low, maintenance dosage. Reduction ofHRT or estrogen can occur by reducing HRT or estrogen by, e.g., 1%, 2%,5%, 10%, 15%, 30%, or 50%, over a period of, e.g., one week, two weeks,one to six weeks, one month, two months, one to six months, two to sixmonths, six months to a year, one year, two years, three years, fouryears and five years.

Gradually decreasing estrogen function can include increasing dosage ofan estrogen antagonist or an estrogen receptor antagonist over a periodof, e.g., one week, two weeks, one to six weeks, one month, two months,one to six months, two to six months, six months to a year, one year,two years, three years, four years and five years. Increasing dosage ofan estrogen antagonist or an estrogen receptor antagonist can occur byincreasing the dosage of estrogen antagonist or an estrogen receptorantagonist by, e.g., 50%, 100%, 200%, 500%, or 1000% over a period of,e.g., one week, two weeks, one to six weeks, one month, two months, oneto six months, two to six months, six months to a year, one year, twoyears, three years, four years and five years.

C. Agents

1. Estrogen, Estrogen Analogs, Estrogen Agonists

Forms of estrogen, estrogen analogs and estrogen agonists suitable foruse with the present invention include estradiol, estradiol acetate,ethinyl estradiol, mestranol, conjugated synthetic estrogens (e.g.,Enjuvia®, which contains sodium delta-8,9-dehydroestrone sulfate, sodiumestrone sulfate, sodium equilin sulfate, sodium 7-alpha-dihydroequilinsulfate, sodium 17-alpha-estradiol sulfate, sodium17-beta-dihydroequilin sulfate, sodium 17-alpha-dihydroequileninsulfate, sodium 17-beta-dihydroequilenin sulfate, sodium equileninsulfate, and sodium 17-beta-estradiol sulfate.), and conjugated equineestrogens (e.g., Premarin®). Estrogen may be administered by any meansknown in the art, including orally, transdermally, vaginally. Estrogendosages suitable for use with the present invention include, forexample, between about 0.01 to about 0.1 mg/day. In certain embodiments,15 μg, 20 μg, 30 μg, or 50 μg estrogen (e.g., ethinyl estradiol) isadministered per day for a given time period (e.g., 21 days), and thenadministration is repeated immediately, or after a week of noadministration.

2. Agents that Antagonize Estrogen Function

One class of agents that antagonize estrogen function suitable for usein the present invention prevents hormones from attaching to cancercells, e.g., Tamoxifen. Tamoxifen is a selective estrogen receptormodulator (SERM). SERMs act by blocking any estrogen present in the bodyfrom attaching to the estrogen receptor on the cancer cells, slowing thegrowth of tumors and killing tumor cells. In certain embodiments, theSERM is clomifene, femarelle, ormeloxifene, raloxifene, toremifene,lasofoxifene, ospemifene, afimoxifene, arzoxifene or bazedoxifene. Incertain embodiments, a selective estrogen receptor down-regulator (SERD)is used. In certain embodiments, the SERD is fulvestrant (Faslodex®).Tamoxifen can be used in both pre- and postmenopausal women.

Another class of agents that antagonize estrogen function suitable foruse in the present invention arrest estrogen production after menopause.For instance, aromatase inhibitors block the action of an enzyme thatconverts androgens into estrogen. Specifically, aromatase inhibitors areeffective only in postmenopausal women, and include commonly knowndrugs, such as, anastrozole (Arimidex), letrozole (Femara) andexemestane (Aromasin).

D. Formulations

Pharmaceutical compositions of the disclosure (e.g., estrogen or agentsthat antagonize estrogen function) may be administered by various means,depending on their intended use, as is well known in the art. Forexample, if compositions of the disclosure are to be administeredorally, they may be formulated as tablets, capsules, granules, powdersor syrups. Alternatively, formulations disclosed herein may beadministered parenterally as injections (intravenous, intramuscular orsubcutaneous), drop infusion preparations or suppositories. Forapplication by the ophthalmic mucous membrane route, the compositionsdisclosed herein may be formulated as eye drops or eye ointments. Theseformulations may be prepared by conventional means, and, if desired, thecompositions may be mixed with any conventional additive, such as anexcipient, a binder, a disintegrating agent, a lubricant, a corrigent, asolubilizing agent, a suspension aid, an emulsifying agent or a coatingagent. The disclosed excipients may serve more than one function. Forexample, fillers or binders may also be disintegrants, glidants,anti-adherents, lubricants, sweeteners and the like.

In formulations of the disclosure, wetting agents, emulsifiers andlubricants, such as sodium lauryl sulfate and magnesium stearate, aswell as coloring agents, release agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants may bepresent in the formulated agents.

Subject compositions may be suitable for oral, nasal (e.g., byinhalation using a dry powder formulation or a nebulized formulation),topical (including buccal and sublingual), pulmonary (including aerosoladministration), rectal, vaginal, aerosol and/or parenteral (e.g., byinjection, for example, intravenous or subcutaneous injection)administration. The formulations may conveniently be presented in unitdosage form and may be prepared by any methods well known in the art ofpharmacy. The amount of a composition that may be combined with acarrier material to produce a single dose vary depending upon thesubject being treated, and the particular mode of administration.

Methods of preparing these formulations include the step of bringinginto association compositions of the disclosure with the carrier and,optionally, one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation agents with liquid carriers, or finely divided solidcarriers, or both, and then, if necessary, shaping the product.

Formulations suitable for oral administration may be in the form ofcapsules, cachets, pills, tablets, lozenges (using a flavored basis,usually sucrose and acacia or tragacanth), powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia), each containing a predetermined amount of a subjectcomposition thereof as an active ingredient. Compositions of thedisclosure may also be administered as a bolus, electuary, or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules and the like), the subject composition ismixed with one or more pharmaceutically acceptable carriers, such assodium citrate or dicalcium phosphate, and/or any of the following: (1)fillers or extenders, such as starches, dextrose, lactose, sucrose,glucose, mannitol, and/or silicic acid; (2) binders, such as, forexample, celluloses (e.g., microcrystalline cellulose, methyl cellulose,hydroxypropylmethyl cellulose (HPMC) and carboxymethylcellulose),alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds; (7) wetting agents, such as, for example, cetylalcohol and glycerol monostearate; (8) absorbents, such as kaolin andbentonite clay; (9) lubricants, such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof; and (10) coloring agents. In the case of capsules,tablets and pills, the compositions may also comprise buffering agents.Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugars, as well as high molecular weight polyethylene glycolsand the like. The disclosed excipients may serve more than one function.For example, fillers or binders may also be disintegrants, glidants,anti-adherents, lubricants, sweeteners and the like.

Formulations and compositions may include micronized crystals of thedisclosed compounds. Micronization may be performed on crystals of thecompounds alone, or on a mixture of crystals and a part or whole ofpharmaceutical excipients or carriers. Mean particle size of micronizedcrystals of a disclosed compound may be for example about 5 to about 200microns, or about 10 to about 110 microns.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin, microcrystalline cellulose, orhydroxypropylmethyl cellulose), lubricant, inert diluent, preservative,disintegrant (for example, sodium starch glycolate or cross-linkedsodium carboxymethyl cellulose), surface-active or dispersing agent.Molded tablets may be made by molding in a suitable machine a mixture ofthe subject composition moistened with an inert liquid diluent. Tablets,and other solid dosage forms, such as dragees, capsules, pills andgranules, may optionally be scored or prepared with coatings and shells,such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. The disclosed excipients may serve morethan one function. For example, fillers or binders may also bedisintegrants, glidants, anti-adherents, lubricants, sweeteners and thelike.

It will be appreciated that a disclosed composition may includelyophilized or freeze dried compounds disclosed herein. For example,disclosed herein are compositions that disclosed compounds crystallineand/or amorphous powder forms. Such forms may be reconstituted for useas e.g., an aqueous composition.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the subject composition, the liquid dosage formsmay contain inert diluents commonly used in the art, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (in particular, cottonseed, groundnut, corn, germ, olive,castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan, cyclodextrins and mixturesthereof.

Suspensions, in addition to the subject composition, may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Formulations for rectal or vaginal administration may be presented as asuppository, which may be prepared by mixing a subject composition withone or more suitable non-irritating excipients or carriers comprising,for example, cocoa butter, polyethylene glycol, a suppository wax or asalicylate, and which is solid at room temperature, but liquid at bodytemperature and, therefore, will melt in the body cavity and release theactive agent. Formulations which are suitable for vaginal administrationalso include pessaries, tampons, creams, gels, pastes, foams or sprayformulations containing such carriers as are known in the art to beappropriate.

Dosage forms for transdermal administration of a subject compositionincludes powders, sprays, ointments, pastes, creams, lotions, gels,solutions, and patches. The active component may be mixed under sterileconditions with a pharmaceutically acceptable carrier, and with anypreservatives, buffers, or propellants that may be required.

The ointments, pastes, creams and gels may contain, in addition to asubject composition, excipients, such as animal and vegetable fats,oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays may contain, in addition to a subject composition,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates and polyamide powder, or mixtures of these substances.Sprays may additionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

Compositions and compounds of the disclosure may alternatively beadministered by aerosol. This is accomplished by preparing an aqueousaerosol, liposomal preparation or solid particles containing thecompound. A non-aqueous (e.g., fluorocarbon propellant) suspension couldbe used. Sonic nebulizers may be used because they minimize exposing theagent to shear, which may result in degradation of the compoundscontained in the subject compositions.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of a subject composition together withconventional pharmaceutically acceptable carriers and stabilizers. Thecarriers and stabilizers vary with the requirements of the particularsubject composition, but typically include non-ionic surfactants(Tweens, pluronics, or polyethylene glycol), innocuous proteins likeserum albumin, sorbitan esters, oleic acid, lecithin, amino acids suchas glycine, buffers, salts, sugars or sugar alcohols. Aerosols generallyare prepared from isotonic solutions.

It should be noted that excipients given as examples may have more thanone function. For example, fillers or binders can also be disintegrants,glidants, anti-adherents, lubricants, sweeteners and the like.

Pharmaceutical compositions of this disclosure suitable for parenteraladministration comprise a subject composition in combination with one ormore pharmaceutically-acceptable sterile isotonic aqueous or non-aqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents. Forexample, provided herein is an aqueous composition that includes adisclosed compound, and may further include for example, dextrose (e.g.,about 1 to about 10 weight percent dextrose, or about 5 weight percentdextrose in water (D5W).

Examples of suitable aqueous and non-aqueous carriers which may beemployed in the pharmaceutical compositions of the disclosure includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate and cyclodextrins. Proper fluidity may be maintained,for example, by the use of coating materials, such as lecithin, by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants.

It will be appreciated that contemplated formulations, such as oralformulations (e.g. a pill or tablet), may be formulated as controlledrelease formulation, e.g., an immediate release formulation, a delayedrelease formulation, or a combination thereof.

In certain embodiments, the subject compounds may be formulated as atablet, pill, capsule or other appropriate ingestible formulation(collectively hereinafter “tablet”). In certain embodiments, atherapeutic dose may be provided in 10 tablets or fewer. In anotherexample, a therapeutic dose is provided in 50, 40, 30, 20, 15, 10, 5 or3 tablets.

E. Methods of Predicting Risk

The invention also features methods of predicting an increased risk ofdeveloping a second, independent breast cancer in a subject. The methodincludes detecting a single nucleotide polymorphism (SNP) at position 4of the let-7 complementary site 6 of KRAS in a patient sample whereinthe presence of said SNP indicates an increased risk of developing asecond, independent cancer in said subject. Specifically the mutationthat is detected is a SNP at position 4 of LCS6 of KRAS of which resultsin a uracil (U) or thymine (T) to guanine (G) conversion. In certainembodiments, the second, independent cancer is breast cancer. In certainembodiments, the subject is a breast cancer patient.

Identification of the mutation indicates an increases risk of developingsynchronous and asynchronous second breast cancer. “Risk” in the contextof the present invention, relates to the probability that an event willoccur over a specific time period, and can mean a subject's “absolute”risk or “relative” risk. Absolute risk can be measured with reference toeither actual observation post-measurement for the relevant time cohort,or with reference to index values developed from statistically validhistorical cohorts that have been followed for the relevant time period.Relative risk refers to the ratio of absolute risks of a subjectcompared either to the absolute risks of low risk cohorts or an averagepopulation risk, which can vary by how clinical risk factors areassessed. Odds ratios, the proportion of positive events to negativeevents for a given test result, are also commonly used (odds areaccording to the formula p/(1−p) where p is the probability of event and(1−p) is the probability of no event) to no-conversion.

“Risk evaluation,” or “evaluation of risk” in the context of the presentinvention encompasses making a prediction of the probability, odds, orlikelihood that an event or disease state may occur, the rate ofoccurrence of the event or conversion from one disease state to another,i.e., from a primary tumor to a metastatic tumor or to one at risk ofdeveloping a metastatic, or from at risk of a primary metastatic eventto a secondary metastatic event or from at risk of a developing aprimary tumor of one type to developing a one or more primary tumors ofa different type. Risk evaluation can also comprise prediction of futureclinical parameters, traditional laboratory risk factor values, or otherindices of cancer, either in absolute or relative terms in reference toa previously measured population.

An “increased risk” is meant to describe an increased probability thatan individual who carries the KRAS-variant develops synchronous orasynchronous second breast cancer, compared to an individual who doesnot carry KRAS-variant. In certain embodiments, a KRAS-variant carrieris 1.5×, 2×, 2.5×, 3×, 3.5×, 4×, 4.5×, 5×, 5.5×, 6×, 6.5×, 7×, 7.5×, 8×,8.5×, 9×, 9.5×, 10×, 20×, 30×, 40×, 50×, 60×, 70×, 80×, 90×, or 100×more likely to develop a synchronous or asynchronous second breastcancer than an individual who does not carry the KRAS-variant.

A subject is preferably a mammal. The mammal can be a human, non-humanprimate, mouse, rat, dog, cat, horse, or cow, but are not limited tothese examples. A subject can be male or female.

The biological sample can be any tissue or fluid that contains nucleicacids. Various embodiments include paraffin imbedded tissue, frozentissue, surgical fine needle aspirations, and cells of the breast,endometrium, ovaries, uterus, or cervix. Other embodiments include fluidsamples such peripheral blood lymphocytes, lymph fluid, ascites, serousfluid, sputum, and stool or urinary specimens such as bladder washingand urine.

Linkage disequilibrium (LD) refers to the co-inheritance of alleles(e.g., alternative nucleotides) at two or more different SNP sites atfrequencies greater than would be expected from the separate frequenciesof occurrence of each allele in a given population. The expectedfrequency of co-occurrence of two alleles that are inheritedindependently is the frequency of the first allele multiplied by thefrequency of the second allele. Alleles that co-occur at expectedfrequencies are said to be in “linkage equilibrium”. In contrast, LDrefers to any non-random genetic association between allele(s) at two ormore different SNP sites, which is generally due to the physicalproximity of the two loci along a chromosome. LD can occur when two ormore SNPs sites are in close physical proximity to each other on a givenchromosome and therefore alleles at these SNP sites will tend to remainunseparated for multiple generations with the consequence that aparticular nucleotide (allele) at one SNP site will show a non-randomassociation with a particular nucleotide (allele) at a different SNPsite located nearby. Hence, genotyping one of the SNP sites will givealmost the same information as genotyping the other SNP site that is inLD.

For screening individuals for genetic disorders (e.g. prognostic orrisk) purposes, if a particular SNP site is found to be useful forscreening a disorder, then the skilled artisan would recognize thatother SNP sites which are in LD with this SNP site would also be usefulfor screening the condition. Various degrees of LD can be encounteredbetween two or more SNPs with the result being that some SNPs are moreclosely associated (i.e., in stronger LD) than others. Furthermore, thephysical distance over which LD extends along a chromosome differsbetween different regions of the genome, and therefore the degree ofphysical separation between two or more SNP sites necessary for LD tooccur can differ between different regions of the genome.

For screening applications, polymorphisms (e.g., SNPs and/or haplotypes)that are not the actual disease-causing (causative) polymorphisms, butare in LD with such causative polymorphisms, are also useful. In suchinstances, the genotype of the polymorphism(s) that is/are in LD withthe causative polymorphism is predictive of the genotype of thecausative polymorphism and, consequently, predictive of the phenotype(e.g., disease) that is influenced by the causative SNP(s). Thus,polymorphic markers that are in LD with causative polymorphisms areuseful as markers, and are particularly useful when the actual causativepolymorphism(s) is/are unknown.

Linkage disequilibrium in the human genome is reviewed in: Wall et al.(2003) NAT REV GENET. 4(8):587-97; Gamer et al. (2003) GENET EPIDEMIOL.24 (1):57-67; Ardlie et al. (2002) NAT REV GENET. 3(4):299-309 (erratumin (2002) NAT REV GENET 3(7):566); and Remm et al. (2002) CURR OPIN CHEMBIOL. 6(1):24-30.

The screening techniques of the present invention may employ a varietyof methodologies to determine whether a test subject has a SNP or a SNPpattern associated with an increased or decreased risk of developing adetectable trait or whether the individual suffers from a detectabletrait as a result of a particular polymorphism/mutation, including, forexample, methods which enable the analysis of individual chromosomes forhaplotyping, family studies, single sperm DNA analysis, or somatichybrids. The trait analyzed using the diagnostics of the invention maybe any detectable trait that is commonly observed in pathologies anddisorders.

F. SNP Genotyping Methods

The process of determining which specific nucleotide (i.e., allele) ispresent at each of one or more SNP positions, such as a SNP position ina nucleic acid molecule disclosed in SEQ ID NO: 11, 12 or 13, isreferred to as SNP genotyping. The present invention provides methods ofSNP genotyping, such as for use in screening for a variety of disorders,or determining predisposition thereto, or determining responsiveness toa form of treatment, or prognosis, or in genome mapping or SNPassociation analysis, etc.

Nucleic acid samples can be genotyped to determine which allele(s)is/are present at any given genetic region (e.g., SNP position) ofinterest by methods well known in the art. The neighboring sequence canbe used to design SNP detection reagents such as oligonucleotide probes,which may optionally be implemented in a kit format. Exemplary SNPgenotyping methods are described in Chen et al. (2003) PHARMACOGENOMICSJ. 3(2):77-96; Kwok et al. (2003) CURR ISSUES MOL. BIOL. 5(2):43-60; Shi(2002) AM J PHARMACOGENOMICS 2(3):197-205; and Kwok (2001) ANNU REVGENOMICS HUM GENET 2:235-58. Exemplary techniques for high-throughputSNP genotyping are described in Marnellos (2003) CURR OPIN DRUG DISCOVDEVEL. 6(3):317-21. Common SNP genotyping methods include, but are notlimited to, TaqMan assays, molecular beacon assays, nucleic acid arrays,allele-specific primer extension, allele-specific PCR, arrayed primerextension, homogeneous primer extension assays, primer extension withdetection by mass spectrometry, pyrosequencing, multiplex primerextension sorted on genetic arrays, ligation with rolling circleamplification, homogeneous ligation, OLA (U.S. Pat. No. 4,988,167),multiplex ligation reaction sorted on genetic arrays,restriction-fragment length polymorphism, single base extension-tagassays, and the Invader assay. Such methods may be used in combinationwith detection mechanisms such as, for example, luminescence orchemiluminescence detection, fluorescence detection, time-resolvedfluorescence detection, fluorescence resonance energy transfer,fluorescence polarization, mass spectrometry, and electrical detection.

Various methods for detecting polymorphisms include, but are not limitedto, methods in which protection from cleavage agents is used to detectmismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al. (1985)SCIENCE 230:1242; Cotton et al. (1988) PNAS 85:4397; and Saleeba et al.(1992) METH. ENZYMOL. 217:286-295), comparison of the electrophoreticmobility of variant and wild type nucleic acid molecules (Orita et al.(1989) PNAS 86:2766; Cotton et al. (1993) MUTAT. RES. 285:125-144; andHayashi et al. (1992) GENET. ANAL. TECH. APPL. 9:73-79), and assayingthe movement of polymorphic or wild-type fragments in polyacrylamidegels containing a gradient of denaturant using denaturing gradient gelelectrophoresis (DGGE) (Myers et al. (1985), NATURE 313:495). Sequencevariations at specific locations can also be assessed by nucleaseprotection assays such as RNase and SI protection or chemical cleavagemethods.

In a preferred embodiment, SNP genotyping is performed using the TaqManassay, which is also known as the 5′ nuclease assay (U.S. Pat. Nos.5,210,015 and 5,538,848). The TaqMan assay detects the accumulation of aspecific amplified product during PCR. The TaqMan assay utilizes anoligonucleotide probe labeled with a fluorescent reporter dye and aquencher dye. The reporter dye is excited by irradiation at anappropriate wavelength, it transfers energy to the quencher dye in thesame probe via a process called fluorescence resonance energy transfer(FRET). When attached to the probe, the excited reporter dye does notemit a signal. The proximity of the quencher dye to the reporter dye inthe intact probe maintains a reduced fluorescence for the reporter. Thereporter dye and quencher dye may be at the 5′ most and the 3′ mostends, respectively, or vice versa. Alternatively, the reporter dye maybe at the 5′ or 3′ most end while the quencher dye is attached to aninternal nucleotide, or vice versa. In yet another embodiment, both thereporter and the quencher may be attached to internal nucleotides at adistance from each other such that fluorescence of the reporter isreduced.

During PCR, the 5′ nuclease activity of DNA polymerase cleaves theprobe, thereby separating the reporter dye and the quencher dye andresulting in increased fluorescence of the reporter. Accumulation of PCRproduct is detected directly by monitoring the increase in fluorescenceof the reporter dye. The DNA polymerase cleaves the probe between thereporter dye and the quencher dye only if the probe hybridizes to thetarget SNP-containing template which is amplified during PCR, and theprobe is designed to hybridize to the target SNP site only if aparticular SNP allele is present.

Preferred TaqMan primer and probe sequences can readily be determinedusing the SNP and associated nucleic acid sequence information providedherein. A number of computer programs, such as Primer Express (AppliedBiosystems, Foster City, Calif.), can be used to rapidly obtain optimalprimer/probe sets. It will be apparent to one of skill in the art thatsuch primers and probes for detecting the SNPs of the present inventionare useful in prognostic assays for a variety of disorders includingcancer, and can be readily incorporated into a kit format. The presentinvention also includes modifications of the Taqman assay well known inthe art such as the use of Molecular Beacon probes (U.S. Pat. Nos.5,118,801 and 5,312,728) and other variant formats (U.S. Pat. Nos.5,866,336 and 6,117,635).

The identity of polymorphisms may also be determined using a mismatchdetection technique, including but not limited to the RNase protectionmethod using riboprobes (Winter et al. (1985) PNAS 82:7575; Meyers etal. (1985) Science 230:1242) and proteins which recognize nucleotidemismatches, such as the E. coli mutS protein (Modrich (1991) Ann. Rev.Genet. 25:229-253). Alternatively, variant alleles can be identified bysingle strand conformation polymorphism (SSCP) analysis (Orita et al.(1989) Genomics 5:874-879; Humphries et al., in Molecular Diagnosis ofGenetic Diseases, R. Elles, ed., pp. 321-340, 1996) or denaturinggradient gel electrophoresis (DGGE) (Wartell et al. (1990) Nucl. AcidsRes. 18:2699-2706; Sheffield et al. (1989) PNAS 86:232-236).

A polymerase-mediated primer extension method may also be used toidentify the polymorphism(s). Several such methods have been describedin the patent and scientific literature and include the “Genetic BitAnalysis” method (WO92/15712) and the ligase/polymerase mediated geneticbit analysis (U.S. Pat. No. 5,679,524). Related methods are disclosed inWO91/02087, WO90/09455, WO95/17676, U.S. Pat. Nos. 5,302,509, and5,945,283. Extended primers containing a polymorphism may be detected bymass spectrometry as described in U.S. Pat. No. 5,605,798. Anotherprimer extension method is allele-specific PCR (Ruano et al. (1989)NUCL. ACIDS RES. 17:8392; Ruano et al. (1991) NUCL. ACIDS RES. 19,6877-6882; WO 93/22456; Turki et al. (1995) J CLINT. INVEST.95:1635-1641). In addition, multiple polymorphic sites may beinvestigated by simultaneously amplifying multiple regions of thenucleic acid using sets of allele-specific primers as described inWallace et al. (WO89/10414).

Another preferred method for genotyping the SNPs of the presentinvention is the use of two oligonucleotide probes in an OLA (see, e.g.,U.S. Pat. No. 4,988,617). In this method, one probe hybridizes to asegment of a target nucleic acid with its 3′ most end aligned with theSNP site. A second probe hybridizes to an adjacent segment of the targetnucleic acid molecule directly 3′ to the first probe. The two juxtaposedprobes hybridize to the target nucleic acid molecule, and are ligated inthe presence of a linking agent such as a ligase if there is perfectcomplementarity between the 3′ most nucleotide of the first probe withthe SNP site. If there is a mismatch, ligation would not occur. Afterthe reaction, the ligated probes are separated from the target nucleicacid molecule, and detected as indicators of the presence of a SNP.

The following patents, patent applications, and published internationalpatent applications, which are all hereby incorporated by reference,provide additional information pertaining to techniques for carrying outvarious types of OLA: U.S. Pat. Nos. 6,027,889, 6,268,148, 5,494,810,5,830,711, and 6,054,564 describe OLA strategies for performing SNPdetection; WO 97/31256 and WO 00/56927 describe OLA strategies forperforming SNP detection using universal arrays, wherein a zipcodesequence can be introduced into one of the hybridization probes, and theresulting product, or amplified product, hybridized to a universal zipcode array; U.S. application US01/17329 (and Ser. No. 09/584,905)describes OLA (or LDR) followed by PCR, wherein zipcodes areincorporated into OLA probes, and amplified PCR products are determinedby electrophoretic or universal zipcode array readout; U.S. application60/427,818, 60/445,636, and 60/445,494 describe SNPlex methods andsoftware for multiplexed SNP detection using OLA followed by PCR,wherein zipcodes are incorporated into OLA probes, and amplified PCRproducts are hybridized with a zipchute reagent, and the identity of theSNP determined from electrophoretic readout of the zipchute. In someembodiments, OLA is carried out prior to PCR (or another method ofnucleic acid amplification). In other embodiments, PCR (or anothermethod of nucleic acid amplification) is carried out prior to OLA.

Another method for SNP genotyping is based on mass spectrometry. Massspectrometry takes advantage of the unique mass of each of the fournucleotides of DNA. SNPs can be unambiguously genotyped by massspectrometry by measuring the differences in the mass of nucleic acidshaving alternative SNP alleles. MALDI-TOF (Matrix Assisted LaserDesorption Ionization—Time of Flight) mass spectrometry technology ispreferred for extremely precise determinations of molecular mass, suchas SNPs. Numerous approaches to SNP analysis have been developed basedon mass spectrometry. Preferred mass spectrometry-based methods of SNPgenotyping include primer extension assays, which can also be utilizedin combination with other approaches, such as traditional gel-basedformats and microarrays.

Typically, the primer extension assay involves designing and annealing aprimer to a template PCR amplicon upstream (5′) from a target SNPposition. A mix of dideoxynucleotide triphosphates (ddNTPs) and/ordeoxynucleotide triphosphates (dNTPs) are added to a reaction mixturecontaining template (e.g., a SNP-containing nucleic acid molecule whichhas typically been amplified, such as by PCR), primer, and DNApolymerase. Extension of the primer terminates at the first position inthe template where a nucleotide complementary to one of the ddNTPs inthe mix occurs. The primer can be either immediately adjacent (i.e., thenucleotide at the 3′ end of the primer hybridizes to the nucleotide nextto the target SNP site) or two or more nucleotides removed from the SNPposition. If the primer is several nucleotides removed from the targetSNP position, the only limitation is that the template sequence betweenthe 3′ end of the primer and the SNP position cannot contain anucleotide of the same type as the one to be detected, or this willcause premature termination of the extension primer. Alternatively, ifall four ddNTPs alone, with no dNTPs, are added to the reaction mixture,the primer will always be extended by only one nucleotide, correspondingto the target SNP position. In this instance, primers are designed tobind one nucleotide upstream from the SNP position (i.e., the nucleotideat the 3′ end of the primer hybridizes to the nucleotide that isimmediately adjacent to the target SNP site on the 5′ side of the targetSNP site). Extension by only one nucleotide is preferable, as itminimizes the overall mass of the extended primer, thereby increasingthe resolution of mass differences between alternative SNP nucleotides.Furthermore, mass-tagged ddNTPs can be employed in the primer extensionreactions in place of unmodified ddNTPs. This increases the massdifference between primers extended with these ddNTPs, thereby providingincreased sensitivity and accuracy, and is particularly useful fortyping heterozygous base positions. Mass-tagging also alleviates theneed for intensive sample-preparation procedures and decreases thenecessary resolving power of the mass spectrometer.

The extended primers can then be purified and analyzed by MALDI-TOF massspectrometry to determine the identity of the nucleotide present at thetarget SNP position. In one method of analysis, the products from theprimer extension reaction are combined with light absorbing crystalsthat form a matrix. The matrix is then hit with an energy source such asa laser to ionize and desorb the nucleic acid molecules into thegas-phase. The ionized molecules are then ejected into a flight tube andaccelerated down the tube towards a detector. The time between theionization event, such as a laser pulse, and collision of the moleculewith the detector is the time of flight of that molecule. The time offlight is precisely correlated with the mass-to-charge ratio (m/z) ofthe ionized molecule. Ions with smaller m/z travel down the tube fasterthan ions with larger m/z and therefore the lighter ions reach thedetector before the heavier ions. The time-of-flight is then convertedinto a corresponding, and highly precise, m/z. In this manner, SNPs canbe identified based on the slight differences in mass, and thecorresponding time of flight differences, inherent in nucleic acidmolecules having different nucleotides at a single base position. Forfurther information regarding the use of primer extension assays inconjunction with MALDI-TOF mass spectrometry for SNP genotyping, see,e.g., Wise et al., “A standard protocol for single nucleotide primerextension in the human genome using matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry”, RAPID COMMUNMASS SPECTROM. 2003; 17 (11):1195-202.

The following references provide further information describing massspectrometry-based methods for SNP genotyping: Bocker (2003)BIOINFORMATICS 19 Suppl 1:144-153; Storm et al. (2003) METHODS MOL.BIOL. 212:241-62; Jurinke et al. (2002) ADV BIOCHEM ENG BIOTECHNOL.77:57-74; and Jurinke et al. (2002) METHODS MOL. BIOL. 187:179-92.

SNPs can also be scored by direct DNA sequencing. A variety of automatedsequencing procedures can be utilized ((1995) BIOTECHNIQUES 19:448),including sequencing by mass spectrometry (see, e.g., PCT InternationalPublication No. WO94/16101; Cohen et al. (1996) ADV. CHROMATOGR.36:127-162; and Griffin et al. (1993) APPL. BIOCHEM. BIOTECHNOL.38:147-159). The nucleic acid sequences of the present invention enableone of ordinary skill in the art to readily design sequencing primersfor such automated sequencing procedures. Commercial instrumentation,such as the Applied Biosystems 377, 3100, 3700, 3730, and 3730xl DNAAnalyzers (Foster City, Calif.), is commonly used in the art forautomated sequencing.

Other methods that can be used to genotype the SNPs of the presentinvention include single-strand conformational polymorphism (SSCP), anddenaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985)NATURE 313:495). SSCP identifies base differences by alteration inelectrophoretic migration of single stranded PCR products, as describedin Orita et al., PROC. NAT. ACAD. Single-stranded PCR products can begenerated by heating or otherwise denaturing double stranded PCRproducts. Single-stranded nucleic acids may refold or form secondarystructures that are partially dependent on the base sequence. Thedifferent electrophoretic mobilities of single-stranded amplificationproducts are related to base-sequence differences at SNP positions. DGGEdifferentiates SNP alleles based on the different sequence-dependentstabilities and melting properties inherent in polymorphic DNA and thecorresponding differences in electrophoretic migration patterns in adenaturing gradient gel (Erlich, ed., PCR Technology, Principles andApplications for DNA Amplification, W. H. Freeman and Co, New York,1992, Chapter 7).

Sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can also be usedto score SNPs based on the development or loss of a ribozyme cleavagesite. Perfectly matched sequences can be distinguished from mismatchedsequences by nuclease cleavage digestion assays or by differences inmelting temperature. If the SNP affects a restriction enzyme cleavagesite, the SNP can be identified by alterations in restriction enzymedigestion patterns, and the corresponding changes in nucleic acidfragment lengths determined by gel electrophoresis

SNP genotyping can include the steps of, for example, collecting abiological sample from a human subject (e.g., sample of tissues, cells,fluids, secretions, etc.), isolating nucleic acids (e.g., genomic DNA,mRNA or both) from the cells of the sample, contacting the nucleic acidswith one or more primers which specifically hybridize to a region of theisolated nucleic acid containing a target SNP under conditions such thathybridization and amplification of the target nucleic acid regionoccurs, and determining the nucleotide present at the SNP position ofinterest, or, in some assays, detecting the presence or absence of anamplification product (assays can be designed so that hybridizationand/or amplification will only occur if a particular SNP allele ispresent or absent). In some assays, the size of the amplificationproduct is detected and compared to the length of a control sample; forexample, deletions and insertions can be detected by a change in size ofthe amplified product compared to a normal genotype.

EXAMPLES Example 1 Summary:

Women with breast cancer (BC) were recruited to join this study, askedto complete a questionnaire on life hormonal exposures, and required tosupply a DNA sample for KRAS-variant testing (n=1712). A group ofKRAS-variant unaffected (cancer free) female controls (n=80) werecollected to compare with KRAS-variant BC patients (n=286). Theassociation of life hormonal exposures with BC was evaluated, as wastumor biology in post-menopausal women with and without a history ofhormone replacement therapy. Isogenic normal breast epithelial celllines with or without the KRAS-variant were used to confirm the impactof estrogen withdrawal on transformation in vitro. The association ofthe KRAS-variant with second primary breast cancer development wasassessed, as were characteristics of presentation.

Compared to non-variant BC patients, KRAS-variant BC patients wentthrough menopause at a significantly younger age (p=0.048), and weremore likely to be on hormone replacement therapy (HRT) when diagnosedwith BC (p=0.032). For women with the KRAS-variant, a history ofdiscontinuation of hormone replacement therapy (HRT) was significantlyassociated with triple negative breast cancer tumor biology and highertumor grade. Compared to KRAS-variant cancer free controls, KRAS-variantBC patients were younger at menopause (p=0.035), had fewer live births(p<0.001), were older at the time of their first birth (p=0.014), weremore likely to have used OCPs (p=0.041), and had a lower body mass index(BMI)(p<0.001). Isogenic breast cell lines with the KRAS-variantexhibited transformation with acute estrogen withdrawal, consistent withthe clinical findings. Most strikingly, homozygous KRAS-variant patients(GG) had over an 11 fold increased risk of developing a second primarybreast cancer compared to non-variant patients (45.39% vs 6.78%, OR11.44 [3.42-37.87], p<0.001). As a group (TG+GG), KRAS-variant BCpatients were over twice as likely to be diagnosed with a second,independent breast cancer than non-variant patients (12.93% vs 6.78%; OR2.04 [1.36-3.06], p<0.001). These findings were significant controllingfor lobular histology, length of follow-up and type of surgery.

Methods Study Groups:

A cohort of breast cancer patients were invited to join a study throughthe Susan Love foundation, called “The KRAS-variant and hormones”(http://www.armyofwomen.org/current/view?grant_id=438). 1906 womenresponded to the invitation and completed questionnaires regarding ageat diagnosis; anthropomorphic measurements including weight and height;reproductive history including parity, age at first birth, use ofcontraceptives and hormone replacement therapy; and personal andfamilial cancer history. Participants signed a consent approved throughthe Yale University Human Investigation Committee (HIC), and were maileda cheek swab or saliva kit (Oragene) for DNA testing, and requested tosupply pathology reports for their BC(s). Pathology reports were used inall cases of second primary breast cancers, where synchronous secondprimary breast cancers were either in the contralateral breast, or if inthe same breast were classified as multi-centric on the pathologyreports, with different pathologies. Metachronous breast cancer was ofdifferent pathology if in the same breast and classified as a newprimary, or in the contralateral breast.

Control samples were provided by the Human Genetics Sample bank at theOhio State University Medical Center (OSUMC). All controls were womenwho had the KRAS-variant but were unaffected by cancer at the time oftesting, n=80. The Columbus Area Controls Sample Bank is a collection ofcontrol samples for use in human genetics research that includes bothdonors' anonymized biological specimens and linked phenotypic data. Thedata and samples are collected under the protocol “Collection andStorage of Controls for Genetics Research Studies”, which is approved bythe Biomedical Sciences Institutional Review Board at OSUMC. Recruitmenttakes place in OSUMC primary care and internal medicine clinics. Ifindividuals agree to participate, they provide written informed consent,complete a questionnaire that includes demographic, medical and familyhistory information, and donate a blood sample, which is used forgenomic DNA extraction and the establishment of an EBV-transformedlymphoblastoid cell culture, cell pellet in Trizol, and plasma.

KRAS-Variant Testing:

For all participants, DNA was extracted from buccal swabs or salivaaccording to the manufacturer's protocol (Oragene). Coded patientsamples were genotyped for the KRAS-variant using a Taqman-based assay,in the MiraDx CLIA certified laboratory, as previously described (Chin,supra).

Isogenic Cell Line Creation:

MCF10a cells are an immortalized, non-transformed mammary epithelialcell line derived from human fibrocystic mammary tissue. These cellshave several characteristics of normal human breast epithelium,including lack of tumorigenicity in nude mice and lack of anchorageindependent growth (Soule et al. (1990) CANCER RES. 50:6075-6086). Thekaryotype of this cell line is near diploid with minimal rearrangement,and they are dependent on exogenous growth factors and hormones forproliferation and survival. They do not express Estrogen Receptor α(ERα). Furthermore, MCF10a cells can form three-dimensional structuresin a reconstituted basement membrane resulting in formation ofpolarized, growth-arrested acini-like spheroids, which are similar toglandular architecture of epithelium in vivo. (Debnath et al. (2003)METHODS 30:256-268).

Isogenic MCF10A lines were generated with and without the KRAS-variantusing the CompoZr™ custom designed zinc-finger nuclease (ZFN) targetedgenome editing technology (Sigma-Aldrich, per manufacturer'sinstructions) (Urnov et al. (2005) NATURE 435(7042):646-51). A ZFN pairwas designed and constructed to specifically target the KRAS 3′UTR. Thedonor construct containing the homology arms on either side of theKRAS-variant was generated by PCR amplifying a 2087 base pair regioncontaining the KRAS-variant from genomic DNA with forward primer 5′AGGACTCTGATTTTGAGGACATC 3′ and reverse primer 5′ AACATGCCCCACAAAGTTTC 3′and cloning into the pGEM-T (Promega) cloning vector. The ZFN plasmids(500 ng) and the donor plasmid (2 μg) were transfected into 2×10⁵ MCF10Acells by nucleofection, program T-024, according to manufacturer'sinstructions (Amaxa), in media containing 100 μM chloroquine. The mediawas changed after 4 hrs and the cells were incubated overnight andre-seeded as single cells into 24-well plates. After passage and DNAcollection, clones were assessed for the presence of the KRAS-variantusing an allele-specific primer and a PCR-based TaqMan assay. Secondaryvalidation was carried out by allele-specific sequencing of TOPO TA®cloned, PCR amplified genomic DNA using forward primer 5′AAGGCATACTAGTACAAGTGGTAATTT 3′ and reverse primer 5′TAGGAGTAGTACAGTTCATGACAAAAA 3′, which hybridize to the KRAS locusoutside of the region corresponding to the donor plasmid recombinationsite. In addition, two positive clones were authenticated usingbi-allelic short tandem repeat (STR) analysis at 16 different genomicloci, yielding 32 diagnostic markers for confirmation. (Genetica DNALaboratories, Inc.) STR analysis confirmed that theMCF10A^(KRAS-variant−/−) (Parental, WT) and the twoMCF10A^(KRAS-variant+/−) (MT) cell lines were (a) identical to the ATCCsSTR profile and (b) identical to each other, except for the presence orabsence of the KRAS-variant.

Real-Time PCR for Epithelial and Mesenchymal Markers:

Total RNA was extracted from 2 independent preparations of cells usingTRIzol using standard procedures. cDNA libraries were generated (induplicate) from 1 μg of total RNA using the iScript cDNA LibrarySynthesis Kit (BioRad). mRNA was analyzed (in triplicate) by qPCR usingiQ SYBR Green SuperMix (BioRad) in reactions containing gene specificprimers (listed below). Reactions were amplified in a HT7900 (AppliedBiosystems) for 10 minutes at 95° C. followed by 40 cycles at 95° C. for15 seconds and 60° for 1 minute. mRNA expression was normalized toBeta-Actin and relative expression was calculated using the delta-deltaCt method. Primers were designed to span exon-intron-exon junctions andproduced amplicons of approximately 500 bp. Primers were synthesized atthe Yale Keck Oligonucleotide Synthesis facility.

Western Blotting:

1.5×106 cells in, log growth phase, were washed with ice cold PBS pH 7.4and lysed with 0.5 ml of SDS Sample buffer (BioRad). Whole cell lysateswere boiled at 95° C. for 5 minutes. Increasing amount of whole celllysate (2, 4 and 8 ul) were loaded on a 4-20% gradient gel (BioRad) forquantification. Proteins were transferred to PVDF membrane and blottedfor using the following antibodies:

Protein Vendor Catalog Number GAPD Abcam [6C5] (ab8245) Vimentin Abcam[V9] (ab8069) Fibronectin Abcam (ab2413) Occludin Abcam [EPR8208](ab167161) E-Cadherin Abcam (ab15148) N-Cadherin Abcam (ab18203) SheepAnti-Mouse GE Healthcare (NA931) IgG HRP Life Sciences DonkeyAnti-Rabbit GE Healthcare (NA934) IgG HRP Life Sciences

Protein was visualized using ECL Western Blotting Detection Reagent (GEHealthcare Life Sciences).

Cell Line and Anchorage Independent Growth Assays:

MCF10A (WT) and (MT) cells were cultured in regular DMEM/F12 medium(Invitrogen) as per the Brugge lab protocol (Debnath et al. (2003)METHODS 30:256-268). 3D Matrigel growth experiments were carried out asper Debnath et al. (Id.) Briefly, eight-well slides (Thermo Scientific)were coated with 40 μl of Growth Factor Reduced (GFR) Matrigel™ (BDBioscience) per well and left to solidify for 20 minutes at 37° C. in ahumidified CO2 incubator. Cell suspension (12,500 cells/ml) in regulargrowth media with 20 ng/ml of EGF was prepared. 2% of GFR Matrigel™ wasadded to the cell suspension. 400 μl of Matrigel™ and cell suspensionmixture (total 5000 cells) was added to each well of a Matrigel™precoated chamber slide. Media containing 20 ng/ml EGF was replacedevery 4-days. Cells were photographed on day 15 at 10× magnification.

Anchorage independent growth was assessed as described previously(Sweasy et al. (2005) PNAS 102:14350-5). After thawing and growing cellsuntil confluence in EGF supplemented media (20 ng/ml), cells were platedinto conditions of study for two passages. For estrogen depletionexperiments phenol red free DMEM/F12 medium (Invitrogen) and 5%charcoal-stripped horse serum (Thermo Fisher) were used, and Tamoxifenor estrogen was added to a final concentration of 1 μM after the firstpassage, as appropriate. To plate, 100 μl of MCF10A (WT) or (MT) cellsat a density of 400,000 cells/ml were mixed with 2 ml of media for thecondition under study containing 2 ml 0.7% noble agar (USB). 1 ml of thecell mixture was added to 1 ml of 1.0% noble agar in a well of a 6-welldish. Cells were fed twice weekly by layering on a 50:50 mixture ofmedia with 0.7% agar for 2 weeks, followed by only media for two-threeadditional weeks. The number of colonies present in each of tenmicroscope fields per well from a total of 3 wells per experiment wascounted to evaluate transformation and is reported as an average of the2 separate MT lines.

MicroRNA Microarray Analysis:

Total RNA was extracted from cells using Trizol as standard. MicroRNAcDNA libraries were generated from total RNA (1 ug) using Megaplex RTPrimers (Human Pool A, Applied Biosystems). MicroRNA expression wasanalyzed using a TaqMan Array Human MicroRNA Card A v2.0 (AppliedBiosystems). Reactions were cycled in a HT7900 (Applied Biosystems) for10 minutes at 95° C. followed by 40 cycles at 95° C. for 15 seconds and60° C. for 1 minute. miRNAs that had a Ct value of >35.0000 wereexcluded from further analysis. Relative expression was calculated usingthe delta-delta Ct method.

Data Analysis:

Data was analyzed using the R environment for statistical computing andgraphics. Continuous data was assessed for normality using Shapiro-Wilktest and parametric or non-parametric tests applied as appropriate.Student t tests were used to compare continuous variables that werenormally distributed and Mann Whitney U test for non-normallydistributed data. Categorical data was analyzed using 2×2 contingencytables (chi-square). In order to assess association between thelikelihood of being diagnosed with a second primary breast cancer andKRAS variants, we used logistic regression and quantified differentialrisk through odds ratios (OR). A similar analysis was replicated toassociate the time from primary diagnosis to diagnosis with a secondprimary breast cancer through the Cox proportional hazard model.Differential timing of second primary cancers was compared throughhazard ratios (HR). In both modelling frameworks, when adjusting forpotential confounders, we selected order and scope of interactioneffects through the Bayesian Information Criterion (BIC). In the Coxproportional hazard model, the assumption of proportionality wasassessed both visually by inspection of Kaplan-Meier survival curves andformally, through the analysis of Schoenfeld residuals (p.val>0.10).

Results KRAS-Variant Versus Non-Variant BC Patients

Of the 1712 patients who supplied DNA samples, 17.4% (n=298) had theKRAS-variant, and 70 (4.0%) had other known genetic mutations associatedwith increased breast cancer risk, including BRCA1, BRCA2 and PTEN. Inthe 1642 women without other mutations, 286 (17.42%) had theKRAS-variant, and 1356 (82.58%) did not. Association of self-reportedestrogen exposures in these BC patients were evaluated to determine ifthere were differences for BC patients with versus those without theKRAS-variant. KRAS-variant BC patients in this cohort had a similar ageof diagnosis (51 years vs 50 years, NS), interval since diagnosis andenrollment (6 years, NS), proportion pre-menopausal (50% vs. 55%),pregnancy rate, birth rate, oral contraceptive (OCP) use, age ofmenopause, HRT use and Body Mass Index (BMI), as non-variant BCpatients. However, by univariate analysis, KRAS-variant BC patients weresignificantly more likely to have had an oophorectomy before their BCdiagnosis (15.5% vs 10.7%, p=0.024) and to be on HRT when diagnosed withbreast cancer (66.3% versus 54.4%, p=0.034) than non-variant BC patients(Table 1). By multivariate analysis, KRAS-variant BC patients continuedto be significantly more likely to have a history of ovarian removal(oophorectomy) pre-diagnosis (OR=1.42, CI 1.03-1.42, p=0.033) (Table 1).In addition, although KRAS-variant patients were not significantly morelikely to have a family history of breast or ovarian cancer thannon-variant BC patients (62.66% vs 64.01%, NS), they were significantlymore likely to have a family history of an individual with multipleprimary cancers than non-variant BC patients (4.98% vs 0.92%, p<0.0001).

TABLE 1 Non-variant KRAS-variant 1356 286 P Test Age at 50 (24-77) 51(24-72) 0.576 Mann diagnosis Whitney U Oophorectomy 145 (10.7%) 44(15.4%) 0.024 X² before diagnosis Interval 6 (0-40) 6 (0-35) 0.142 Mannbetween Whitney U diagnosis and enrollment Age at 49 (25-59) 48 (27-57)0.07 Mann menopause Whitney U Menopausal Pre 748 (55%) 142 (50%) 0.089X² status¹ Post 608 (45%) 144 (50%) HRT use² Yes 401 (67%) 104 (73%)0.192 X² No 195 (33%) 39 (27%) On HRT at Yes 217 (54%) 68 (66%) 0.034 X²diagnosis³ No 182 (46%) 35 (34%) Duration of 60 (2-384) 66 (1-444) 0.586Mann HRT³ Whitney U Pregnancy Nulliparous 304 62 0.801 X² ≥1 pregnancy1046 222 Number of 2 (1-11) 2 (1-9) 0.614 Mann pregnancies⁴ Whitney UNumber of 2 (0-7) 2 (0-5) 0.218 Mann live births⁴ Whitney U Age at first27 (15-45) 26 (16-39) 0.085 Mann birth Whitney U OCP Yes 1155 251 0.280X² No 199 35 Duration 7.75 (0.08-35) 7.5 (0.1-40) 0.971 Mann (yrs)Whitney U BMI 24.38 (15.73-64.36) 24.27 (18.01-46.98) 0.824 Mann WhitneyU ¹At diagnosis, cases; at sampling, controls ²For post-menopausal womenonly ³For women on HRT only ⁴Excluding nulliparous women Table 1:KRAS-variant breast cancer versus non-variant breast cancer patients.Women with breast cancer with the KRAS-variant were younger at the ageof menopause, and were more likely to be on hormone replacement therapy(HRT) at the time of diagnosis, compared to non-variant breast cancerpatients.

TABLE 2 BC Patient Characteristics with versus without the KRAS-VariantOR (95% C.I.)[p.val] Baseline Prob = 15.88% Lobular 0.82 (0.53, 1.26)[0.365] ER positive 0.76 (0.52, 1.11) [0.154] Ovaries removed 1.42(1.03, 1.96) [0.033] BMI 0.98 (0.96, 1.01) [0.277] BCP 1.23 (0.78, 1.94)[0.364] Personal Cancer History 0.80 (0.54, 1.20) [0.278] Age atDiagnosis 1.00 (0.98, 1.03) [0.705] Menopause at Diagnosis 1.27 (0.83,1.96) [0.266] Ever pregnant 0.93 (0.65, 1.32) [0.686] Table 2.KRAS-variant BC cases compared to non-variant BC cases.By a logistic regression model, with predictors included in the modelassuming a linear additive structure, BC patients with the KRAS-variantwere more likely to have had an oophorectomy compared to non-variantbreast cancer patients

The Association of HRT Breast Cancer Subtype and Grade

Because of the significant association of HRT use in KRAS-variant BCpatients in the cohort, and a prior report of altered tumor biology andhigher grade for KRAS-variant HRT users (Cerne, supra), association ofHRT use and histologic BC tumor subtype (ER/PR+, HER2+, or ER/PR/HER2−[triple negative]) and grade was evaluated. Post-menopausally diagnosedBC patients were divided into three HRT use groups based on their HRTuse at the time of their diagnosis. These groups comprised “neverusers,” “current users” (women on HRT at the time of the BC diagnosis),or “past HRT users” (women with a history of HRT use preceding their BCdiagnosis by at least 6 months). Histologic BC tumor subtypes forKRAS-variant (n=133) were then compared to non-variant BC patients(n=612) for those with complete histologic tumor information.

Overall, there was no difference in tumor grade between KRAS-variant BCversus non-variant patients, but triple negative breast cancer (TNBC)tumor subtype was significantly more common in women with theKRAS-variant (p=0.029). The correlation between HRT use and histologicBC tumor subtype and grade was then studied. For non-variant BCpatients, there were no differences in the proportion of women with eachtumor subtype between the never, current or past HRT user groups.However, there was a trend for current or past HRT users to have lowergrade breast tumors than never users in agreement with prior reports(Calle, supra), but this difference was not statistically significant.In KRAS-variant BC patients, there were no statistically significantdifferences in tumor subtype or grade between KRAS-variant never andcurrent HRT users. However, past HRT users were significantly morelikely to have TNBC than KRAS-variant never or current HRT BC patients(35.5% [n=11/31] versus 6.6% [n=6/91], p<0.0001). In addition, comparedto non-variant past HRT users, KRAS-variant past HRT users weresignificantly more likely to have TNBC (35.5% versus 7.3% [n=11/151],p<0.0001, Table 3), and had significantly higher grade tumors (2.33versus 1.98, p=0.029).

TABLE 3 KRAS-variant Non-KRAS-variant P value Never on ER+ 77.1% (27/35)85.2% (127/149) NS HRT HER2+ 22.9% (8/28) 19.9% (28/141) NS TN 11.4%(4/35) 9.3% (14/150) NS Grade 2.24 2.16 NS On HRT ER+ 85.5% (47/55)84.8% (156/184) NS when HER2+ 16.3% (7/43) 12.0% (17/142) NS diagnosedTN 3.6% (2/56) 6.6% (12/182) NS (current) Grade 2.02 2.01 NS Stopped ER+53.1% (17/32) 89.7% (139/155) <0.0001 HRT before HER2+ 6.9% (2/29) 11.2%(15/134) NS diagnosis TN 35.5% (11/31) 7.3% (11/151) <0.0001 (past)Grade 2.33 1.98 p = 0.029 Table 3. Histologic breast cancer subtype andhistory of hormone replacement therapy use.Tumor grade between all KRAS-variant versus non-KRAS-variant patientswas non-significant. KRAS-variant patients were significantly morelikely to have triple negative breast cancers as a group (p=0.029).KRAS-variant patients with a history of past HRT use were significantlymore likely to have TNBC than never or current HRT users with theKRAS-variant. KRAS-variant past HRT users were significantly more likelyto have higher grade and TNBC than non-variant past users. There were nodifferences in cancer subtype by HRT use for non-variant patients.

KRAS-Variant BC Patients Versus Controls

We then evaluated if differences in hormonal exposures might impact BCrisk in women with the KRAS-variant, by comparing hormonal exposures inKRAS-variant BC patients (n=286) with a cohort of KRAS-variant cancerfree unaffected controls (n=80). In univariate analysis we foundnumerous significant differences, including factors associated with HRTuse, pregnancy, OCP use and BMI (Table 4). By multivariate analysis weconfirmed that KRAS-variant BC patients remained significantly morelikely to have a lower BMI, and have fewer live births than KRAS-variantcancer free controls (Table 5). Of note, we found no difference in ageof diagnosis vs age of enrollment between the BC patients and thecontrols.

TABLE 4 Control Case N 80 286 Age at (Range/years) 50 (32-58) 48 (27-57)0.037 Mann menopause Whitney U HRT use Yes 18 (43%) 104 (73%) <0.001 X²(post-menopausal No 24 (57%) 39 (27%) at dx/sampling only) Median 84(6-360) 60 (1-444) 0.769 Mann duration of Whitney U HRT (post-menopausal at dx) Pregnancy Nulliparous 10 (12.5%) 62 (22%) 0.064 X² ≥1pregnancy 70 (87.5%) 222 (78%) Number of 3 (1-7) 2 (1-9) 0.194 Mannpregnancies * Whitney U Number of 3 (0-7) 2 (0-5) <0.001 Mann livebirths* Whitney U Median age 24 (16-38) 26 (16-39) 0.009 Mann at firstbirth Whitney U OCP Yes 63 (79%) 251 (88%) 0.041 X² No 17 (21%) 35 (12%)Duration 5 (0.5-28) 7.5 (0.1-40) 0.182 Mann (years) Whitney U BMI 29(17.6-75.3) 24 (18.0-47.0) <0.001 Mann Whitney U *Excluding nulliparouswomen Table 4. KRAS-variant breast cancer patients (cases) versusunaffected KRAS-variant controls.Women with breast cancer with the KRAS-variant have several significantdifferences in hormonal exposures when compared to cancer freeKRAS-variant women.

TABLE 5 Age at diagnosis/enrollment Age at diagnosis Odds Ratio (95% CI)p-value Use of HRT1 0.95 (0.89-1.03) 0.211 Duration of HRT use 2.73(0.91-8.18) 0.07 Number of live births 1.00 (0.99-1.00) 0.62 Age atfirst birth 0.62 (0.39-0.98) 0.04 BMI 1.11 (0.99-1.24) 0.06 OCP use20.93 (0.87-1.00) 0.04 Duration of OCP 2.15 (0.63-7.42) 0.22 Oophorectomybefore 1.01 (0.94-1.09) 0.77 diagnosis/enrollment3 1.3 (0.44-3.89) 0.631Compared to no HRT use. 2Compared to no OCP use. 3Compared to noovarian procedure. Table 5. KRAS-variant BC cases compared toKRAS-variant controlsWomen with breast cancer with the KRAS-variant by a binary logisticmodel were significantly more likely to have fewer live births, and tohave a lower Body Mass Index (BMI)

Estrogen Withdrawal and Transformation in KRAS-Variant MCF10A Cell Lines

Based on the findings that a low estrogen state was associated with BCfor women with the KRAS-variant, the hypothesis that estrogen withdrawalcould lead to increased BC risk was tested. Isogenic MCF10a lines werecreated with (MCF10a^(KRAS+/−), MT1 and MT2) versus without(MCF10a^(KRAS−/−), WT) the KRAS-variant. When grown under standardconditions, MT lines exhibited a mesenchymal spindle phenotype,suggesting a baseline epithelial to mesenchymal transition (EMT) in thepresence of the KRAS-variant FIG. 1A). Consistent with this alteredphenotype, a panel of epithelial and mesenchymal markers measured bymRNA and Western Blot Analysis were consistent with MT lines havingundergone EMT and being more mesenchymal, with significantly lowerE-Cadherin and Occludin, and significantly higher Fibronectin andVimentin than the WT line (FIG. 1B). Furthermore, in 3D culture, MTcells formed small irregular shaped spheroids, whereas WT cells formedwell-organized, polarized spheroids, supporting a mesenchymal phenotypefor MT lines (FIG. 1C) (Debnath, supra). Potentially explaining the EMT,miRNA expression was significantly different between the MT and WTcells, with miR-200c being most dramatically down-regulated miR in theMT cells (FIG. 1D). In addition, we found that KRAS mRNA was lower inthe MT cells, but KRAS protein was slightly elevated (FIG. 2).

Next, WT and MT lines were plated in soft agar to test fortransformation, as measured by anchorage independent growth. There wasno colony formation seen in the presence of EGF during the course of theexperiment for the WT lines or the MT lines, indicating that neither, atbaseline, was transformed (FIG. 3). However, when the lines were grownwithout EGF, as is standard to promote transformation, the WT linesformed no colonies, while the MT lines had colony formation by thesecond soft agar plating, indicating a significantly enhanced tumorinitiation phenotype. The impact of estrogen withdrawal on thistransformation was next evaluated by the use of charcoal stripped serum,tamoxifen, or a combination of the two. In both MUT cell lines, a 2-foldincreased colony formation rate was seen in the presence of Tamoxifen, a6.2 fold increased colony formation rate in charcoal stripped media, anda 7.9 fold increased colony formation rate with the combination(p<0.001, FIG. 1E). Supporting that the impact of charcoal stripping ontransformation was due to estrogen depletion, return of estrogen to themedia resulted in decreased transformation for the MT cell lines(p=0.018, FIG. 4). These findings biologically confirm wide spreadtransformation in normal breast epithelium with acute estrogenwithdrawal in breast cells with the KRAS-variant.

Next, different forms of estrogen inhibitors were tested to determinetheir effects on transformation in the presence of the KRAS-variant.Tamoxifen (ER antagonist in breast), anastrozole (aromatase inhibitor,inhibiting estrogen synthesis), and fulvestrant (a complete ERantagonist) were administered to parental (WT) or two KRAS-variant (L1and L2) MCF10A cell lines and anchorage independent growth was assessedusing the colony formation assay described above. Tamoxifen led to thehighest level of transformation, with almost 10 times more coloniesformed than the anastrozole or fulvestrant treatments. In contrast,estrogen supplementation prevented transformation (FIG. 5). Theseresults show that certain estrogen-inhibiting agents may be more likelyto transform breast epithelial cells in KRAS-variant cells, andtherefore may be contraindicated for KRAS-variant BC patients.

Multiple Primary Breast Cancer Risk in KRAS-Variant BC Patients

Based on the findings that estrogen exposures before the first BCdiagnosis appeared to increase BC risk for women with the KRAS-variant,and because estrogen withdrawal is often a goal of breast cancertreatment, the risk and timing of multiple primary breast cancer wasevaluated in these women. Women with the KRAS-variant (GT or GG)exhibited a 2.04-fold increase in the odds of having a second primaryBC, when compared to women without the variant (p<0.001). For the firsttime, the inventors of the instant application discovered a genetic doseeffect of the KRAS-variant, with women heterozygous (GT) for theKRAS-variant exhibiting a 1.81-fold increase in the odds of having asecond primary BC (p=0.006), and women homozygous for the KRAS-variantexhibiting an 11.64-fold increase in the odds of having second primaryBC (p<0.001), compared to non-variant BC patients. (Table 6.)

Next, it was determined whether second breast cancer for ERAS-variantpatients occurred at the same time as their first diagnosis (synchronousMPBC), or if they continued to be at an elevated risk of a second breastcancer after their first diagnosis (metachronous MPBC). Women with theKRAS-variant exhibited a 2.63-fold increase in the odds of beingdiagnosed with a synchronous second primary BC, when compared to womenwithout the KRAS-variant (6.79% vs. 2.70% with synchronous MPBC,p=0.001). This was again most pronounced for women homozygous for theKRAS-variant, who had a 12.03-fold increased odds of having asynchronous second primary BC compared to non-variant patients (25.02%with synchronous MPBC, p=0.003). Women with the KRAS-variant continuedto be at an elevated risk for a metachronous breast cancer, with a1.72-fold increase in the odds of developing a second primary tumor(8.05% vs 4.84% with metachronous MPBC, p=0.05) when compared tonon-variant patients. This difference was primarily explained by thelarge increase in the odds of metachronous BC for the homozygousKRAS-variant group, who exhibited a 14.72-fold increase in the odds ofdeveloping a second primary BC after their first BC diagnosis (p<0.001).(Table 6, FIG. 6).

TABLE 6 Second Primary Tumor Risk KRAS- Variant Genotype No. % SecondPrimary BC (95% C.I.) OR (95% C.I.) [p.val] TT 1357 6.78% (5.56%-8.25%)1.00 (Baseline) TG or GG 286 12.93% (9.52%-17.32%) 2.04 (1.36-3.06)[<0.001] TG 275 11.64% (8.35%-15.99%) 1.81 (1.18-2.77) [0.006] GG 1145.39% (20.25%-73.04%) 11.44 (3.42-37.87) [<0.001] Synchronous SecondPrimary Tumors KRAS % Second Primary BC (95% C.I.) {OR} [p.val] VariantsNo. Combined Unilateral Contralateral TT 1561 2.70% (1.95%-3.73%) 0.23%(0.07%-0.72%) 2.62% (1.87%-3.64%) {1.00-baseline} {1.00-baseline}{1.00-baseline} TG or GG 1296 6.79% (4.31%-10.50%) 4.49% (2.58%-7.71%)3.00% (1.51%-5.90%) {2.63} [0.001] {20.29} [<0.001] {1.15} [0.73] TG 2576.23% (3.85%-9.87%) 3.85% (2.09%-6.98%) 3.09% (1.55%-6.04%) {2.39}[0.005] {17.22} [<0.001] {1.19} [0.67] GG 8 25.02% (6.27%-62.24%) 30.03%(10.05%-62.01%) 10.02% (1.39%-46.57%) {12.03} [0.003] {184} [<0.001]{4.15} [0.18] Metachronous Second Primary Tumors (Excluding doublemastectomy cases) KRAS % Second Primary BC (95% C.I.) OR [p.val]Variants No. Combined Unilateral Contralateral TT 1393 4.84%(3.74%-6.23%) 0.52% (0.23%-1.14%) 4.40% (3.36%-5.76% {1.00-baseline}{1.00-baseline} {1.00-baseline} TG or GG 236 8.05% (5.19%-12.33%) 2.10%(0.88%-4.97%) {1.72} [0.04} {4.12} [0.02] TG 229 6.98% (4.32%-11.09%)1.73% (0.65%-4.52%) 6.73% (4.16%-10.73%) {1.48} [0.16] {3.38} [0.06]{1.57} [0.13] GG 7 42.80% (14.32%-76.88%) 22.23% (5.57%-57.88%) {14.72}[<0.001] {54.8} [<0.001] Table 6. Second breast cancer risk inKRAS-variant breast cancer patients.Women with the KRAS-variant are significantly more likely to develop asynchronous or metachronous second primary breast cancer, especiallyhomozygous (GG) patients.

MPBC risk for KRAS-variant patients was evaluated, controlling forextent of surgery and time of follow up. KRAS-variant and non-variantpatients did not significantly differ in their choice of lumpectomy,unilateral mastectomy or bilateral mastectomy at the time of diagnosis.(Table 7A & 7B). Controlling for extent of primary surgery, women withthe KRAS-variant who had a unilateral mastectomy were significantly morelikely to have a synchronous second primary tumor (OR=18.42,CI=3.88-87.82, p<0.001) in the breast when compared to non-variantunilateral mastectomy patients. In addition, controlling for number ofyears at risk, women with the KRAS-variant treated with a lumpectomywere significantly more likely to develop a second, metachronous primarybreast cancer (OR=1.84, CI-1.03-3.27, p=0.04) when compared tonon-variant patients treated in the same manner (Table 8).

Table 7

TABLE 7A Frequencies of unilateral and bilateral mastectomy by KRASvariant KRAS % Unilateral (95% C.I.) % Bilateral (95% C.I.) Variant No.OR [p.val] OR [p.val] TT 1352 22.86% (20.70%, 25.16%) 14.13% (12.37%,16.09%) {1.00-baseline} {1.00-baseline} TG or 284 23.60% (19.03%,28.89%) 13.02% (9.59%, 17.45%) GG {1.04} [0.78] {0.91} [0.63]

TABLE 7B Frequencies of second primary BC by Extent of surgery CombinedSynchronous and Metachronous second Primary Tumors % Second Primary BCSurgery No. (95% C.I.) OR (95% C.I.) [p.val] Lumpectomy 1081 6.66%(5.32%, 8.31%) 1.00-Baseline Unilateral 327 5.50% (3.49%, 8.57%) 0.82(0.48, 1.38) [0.45] Bilateral 228 16.66% (12.36%, 22.06%) 2.80 (1.84,4.27) [<0.001] Synchronous Second Primary Tumors % Second Primary BC(95% C.I.) {OR} [p.val] Surgery No. Combined Unilateral ContralateralLumpectomy 1016 0.79% (0.39%, 1.565) 0.20% (0.05%, 0.78%) 0.69% (0.33%,1.44%) {1.00-baseline} {1.00-baseline} {1.00-baseline} Unilateral 3183.13% (1.68%, 5.69%) 3.13% (1.69%, 5.685) 0.31% (0.04%, 2.16%) {4.08}[0.003] {16.38} [<0.001] {0.45} [0.45] Bilateral 228 16.59% (12.34%,21.955) 1.75% (0.65%, 457%) 15.29% (11.16%, 20.57%) {25.11} [0.001]{9.03} [0.01] {26.04} [<0.001] Metachronous Second Primary Tumors(Excluding double mastectomy cases) % Second Primary BC Surgery No. (95%C.I.) OR (95% C.I.) [p.val] Lumpectomy 1074 6.05% (4.78%, 7.65%)1.00-Baseline Unilateral 318 2.84% (1.48%, 5.35%) 0.45 (0.22, 0.92)[0.03]

TABLE 8 Synchronous Second Primary Tumors KRAS TT KRAS TG/GG % SecondPrimary BC OR (95% C.I.) No. (95% C.I.) [p.val] Lumpectomy 1016 0.58%(0.24%, 1.39%) 4.32 (1.15, 16.40) [0.03] Unilateral 318 0.79% (0.20%,3.12%) 18.42 (3.88, 87.82) [<0.001] Bilateral 228 16.13% (11.59%,22.05%) 1.34 (0.56, 3.20) [0.45] Metachronous Second Primary Tumors(Adjusted by no. of years at risk) KRAS TT KRAS TG/GG % Second PrimaryBC OR (95% C.I.) No. (95% C.I.) [p.val] Lumpectomy 1074 5.18% (3.89%,6.86%) 1.84 (1.03, 3.27) [0.04] Unilateral 318 2.52% (1.20%, 5.25%) 2.18(0.62, 7.72) [0.23] Time to Second Primary Tumor Recurrence KRAS TT KRASHazard Ratio (95% C.I.) Hazard Ratio (95% C.I.) No. [P-val] [p.val]TG/GG Lumpectomy 1074 1.00-baseline 1.74 (0.99, 3.07) [0.05] Unilateral318 0.56 (0.18, 0.97) 1.43 (0.36, 5.74) [0.04] [0.61] Odds Ratios (OR)and Hazard Ratios (HR) refer to a comparison of KRAS variants withinextent of surgery category

The association of lobular histology with the KRAS-variant and second BCrisk was evaluated, and it was found that the KRAS-variant was notassociated with lobular histology, although in agreement with priorreports, lobular histology was associated with increased rates of secondprimary BC, both synchronous and metachronous (Table 9A & 9B).Controlling for lobular histology, extent of surgery and number of yearsat risk, women with the KRAS-variant who had a unilateral mastectomywere significantly more likely to have a synchronous second primarytumor (OR=40.75, CI=4.98-339.72, p<0.01). In addition, women with theKRAS-variant treated with lumpectomy and with non-lobular histologycontinued to be significantly more likely to develop a second,metachronous primary breast cancer (OR=2.01, CI=1.05-3.86, p=0.04).Similar conclusions were found using a time to event analysis (HR=2.01,p=0.03)(Table 10).

To confirm that having the KRAS-variant was an independent predictor ofMPBC, we performed a multivariate analysis using a logistic regressionmodel, assuming that the predictors included in the model had a linearadditive structure. We confirmed using this model that the KRAS-variantwas an independent predictor of MPBC risk considering all other riskfactors (OR=2.26, CI 1.44-2.26, P<0.001, Table 11).

Table 9

TABLE 9A Combined Synchronous and Metachronous second Primary TumorsKRAS Variant No. % Lobular (95% C.I.) OR (95% C.I.) [p.val] TT 118315.30% (13.36%, 17.46%) 1.00 (Baseline) TG or GG 249 12.45% (8.89%,17.13%) 0.79 (0.52, 1.18) [0.25] TG 239 12.55% (8.92%, 17.41%) 0.79(0.52, 1.20) [0.28] GG 10 9.96% (1.39%, 46.46%) 0.61 (0.08, 4.83) [0.65]

TABLE 9B Frequencies of second primary BC by Lobular Status CombinedSynchronous and Metachronous second Primary Tumors % Second Primary BCNo. (95% C.I.) OR (95% C.I.) [p.val] Non 1220 7.79% (6.41%, 9.42%) 1.00(Baseline) Lobular Lobular 212 13.68% (9.67%, 18.99%) 1.88 (1.20, 2.93)[0.006] Synchronous Second Primary rumors % Second Primary BC No. (95%C.I.) OR (95% C.I.) [p.val] Non 1663 3.27% (2.39%, 4.46%) 1.00(Baseline) Lobular Lobular 196 6.63% (3.89%, 11.10%) 2.10 (1.10, 4.03)[0.02] Metachronous Second Primary Tumors (Excluding double mastectomycases) % Second Primary BC No. (95% C.I.) OR (95% C.I.) [p.val] Non 10415.48% (4.25%, 7.03%) 1.00 (Baseline) Lobular Lobular 156 10.27% (6.39%,16.12%) 1.97 (1.10, 3.54) [0.02]

TABLE 10 Frequencies of second primary BC by Extent of Surgery,Histology and Time Synchronous Tumors KRAS TT KRAS TG/GG % SecondPrimary BC OR (95% C.I.) No. (95% C.I.) [p.val] Lumpectomy Non- 7480.51% (0.19%-1.36%) 4.43 (0.97-20.38) [>0.5] Lobular Lobular 97 0.94%(0.29%-2.95%) 2.88 (0.40-20.99) [>0.5] Unilateral Non- 247 0.38%(0.05%-2.67%) 40.75 (4.98-339.72) [<0.01] Lobular Lobular 45 0.70%(0.09%-5.20%) 26.55 (2.42-295.05) [<0.01] Bilateral Non- 166 13.62%(8.96%-20.15%) 6.44 (0.98-41.97) [>0.5] Lobular Lobular 54 22.57%(12.93%-36.29%) 0.95 (0.24-3.75) [>0.5] Metachronous Second PrimaryTumors (Adjusted by no. years at risk) KRAS TT KRAS TG/GG % SecondPrimary BC OR (95% C.I.) No. (95% C.I.) [p.val] Lumpectomy Non- 7924.89% (3.49%-6.80%) 2.01 (1.05-3.86) [0.04] Lobular Lobular 110 12.08%(7.23%-19.46%) 1.08 (0.21-5.38) [>0.5] Unilateral Non- 245 1.77%(0.76%-4.06%) 1.54 (0.29-8.27) [>0.5] Lobular Lobular 47 4.59%(1.86%-10.82%) 0.83 (0.10-6.92) [>0.5] Time to Second Primary TumorRecurrence KRAS TT KRAS TG/GG HR (95% C.I.) HR (95% C.I.) No. [p.val][p.val] Lumpectomy Non- 792 1.00-Baseline 2.01 (1.08-3.77) [0.03]Lobular Lobular 110 2.39 (1.31-4.36) [<0.001] 1.38 (0.30-6.21) [>0.5]Unilateral Non- 245 0.34 (0.15-0.81) [<0.001] 1.31 (0.26-6.69) [>0.5]Lobular Lobular 47 0.82 (0.30-2.29) [>0.5] 0.89 (0.11-7.23) [>0.5] OddsRatios (OR) and Hazard Rations (HR) refer to a comparison ofKRAS.S-variants within extent of surgery category and Lobular statusTable 10. Second breast cancer risk in KRAS-variant breast cancerpatients controlling for lobular histology, extent of surgery and time.Women with the KRAS-variant continue to be at a significantly increasedrisk of synchronous and metachronous breast cancer when controlling forlobular histology, extent of surgery and time.

TABLE 11 Multivariable analysis of multiple cancer risk OR (95% C.I.)[p.val] Baseline Prob = 8.78% KRAS 2.26 (2.26, 1.44) [<0.001] Mastectomy(Unilateral) 0.56 (0.56, 0.30) [0.071] Mastectomy (Bilateral) 2.54(2.54, 1.58) [<0.001] Lobular 1.64 (1.64, 0.99) [0.054] ER positive 0.87(0.87, 0.50) [0.623] Ovaries removed 1.30 (1.30, 0.84) [0.243] BMI 1.00(1.00, 0.96) [0.947] BCP 1.10 (1.10, 0.60) [0.758] Personal CancerHistory 1.29 (1.29, 0.78) [0.322] Age at Diagnosis 1.04 (1.04, 1.01)[0.010] Menopause at Diagnosis 0.40 (0.40, 0.22) [0.003] Ever pregnant0.91 (0.91, 0.56) [0.698] Table 11: The KRAS-variant is an independentpredictor of multiple primary breast cancer in a multivariable model.Results are based on a logistic regression model, and predictors areincluded in the model assuming a linear additive structure.

The results described herein indicate that estrogen withdrawal increasesBC risk in women with the KRAS-variant, who are also significantly morelikely to present with and develop MPBC. This finding was confirmedbiologically in cell lines with the ERAS variant compared to isogeniccontrols. Accordingly, the present invention provides methods foridentifying subject with a KRAS-variant and for preventing cancer byadministering to the KRAS-variant subject an amount of estrogeneffective to reduce the risk of developing cancer, and for treatingcancer by gradually decreasing estrogen exposure in a KRAS-variantsubject to reduce the risk of aggressive tumor growth.

The results described herein also indicate that BC risk for women withthe KRAS-variant appears to be increased when they are in a low estrogenstate, and that abrupt estrogen withdrawal, as found with oophorectomy,discontinuation of HRT, or in the cell line assays described herein,enhances transformation and can increase their risk of aggressive tumorbiology. In addition, women with the KRAS-variant are significantly morelikely to have multiple primary synchronous BC, although also continueto be at risk of metachronous BC development. Further, homozygousKRAS-variant mutant patients are at significantly higher risk thanheterozygotes.

The role of estrogen withdrawal on BC risk for women with theKRAS-variant could be due to a relationship between the KRAS-variant,its downstream pathways and estrogen signaling, as there are knowninteractions between estrogen signaling and the RAS pathway.Alternatively, the relationship between estrogen and the KRAS-variantmay instead be due to alterations in miRNA expression or regulationcaused by this powerful hormone. In support of the later, we havepreviously shown that TNBC tumors from women with the KRAS-variant havesignificantly higher aromatase expression and ER Beta expression. Bothof these genes are regulated by the miRNA let-7, which is known to below in KRAS-variant associated tissues and tumors. One could speculatethat sudden estrogen withdrawal disrupts these biological interactionsin KRAS-variant tissues, ultimately leading to escape, independentsignaling and growth, and oncogenesis. Regardless, our cell linefindings confirm that breast cells with the KRAS-variant are transformedby estrogen withdrawal. In addition, our clinical findings that BCpatients with the KRAS-variant are more likely to have an oophorectomythan non-variant patients, have a lower BMI, and thus lower circulatingestrogen than controls, and that HRT discontinuation leads to aggressivetumor biology, supports the hypothesis that acute estrogen withdrawalalters breast cell biology for KRAS-variant individuals.

A genetic marker of increased risk of synchronous multiple primarybreast cancer has not been previously identified. Other breast cancerassociated genetic mutations are generally considered to predict anincreased risk of second metachronous breast cancers, likely due to thecontinued DNA damage-prone state of the tissues in these individuals.Women with the KRAS-variant are at highest risk of presenting withsynchronous multiple primary cancers, suggesting perhaps that there wasan initiating “event” that promoted cancer initiation, and that theirtissues were globally impacted. Their increased risk of metachronous BCmay be due to treatment for their first breast cancer, as BC treatmentsgeneral involve estrogen withdrawal.

The findings described herein further highlight the critical importanceof studying 3′UTR miRNA binding site mutations in the appropriatecontext. Simply put, 3′UTR mutations should be viewed as an entirelydifferent paradigm of cancer causing mutation. Instead of being amutation that impacts appropriate DNA repair, like BRCA mutations, theyare mutations that alter the appropriate cellular response to externalfactors. Therefore, the general strategy to increase patient numbers bycombining patients of numerous ethnic backgrounds and cultures isclearly a poor solution when studying such 3′UTR mutations. As theselarge consortia have begun to study these mutations, it should not beignored that their findings may be biased against mutations that areperhaps the most important—mutations that might be altered, orcontrolled, by lifestyle modifications. Utilizing the appropriatecohorts to define the factors that can modify cancer risk in meaningful3′UTR mutations should be an extremely high priority in cancerprevention studies at this time.

Limitations of the studies described herein include self-reportedlifestyle factors for our BC patients, which are prone to recall bias.However, our findings regarding the impact of estrogen withdrawal onKRAS-variant tissues were biologically confirmed in cell lines, furthervalidating our results. In addition, some of the strongest findings,regarding tumor biology post-HRT, and second breast cancer risk, wereall confirmed with pathology documentation. Another limitation of thestudy is that the population was not prospectively collected, allowingsurvivor bias for metachronous BC development. However, the cohorts inthis study have identical length of follow up, and we controlled fortime in our metachronous breast cancer analysis. Also, as KRAS-variantwomen are significantly more likely to be diagnosed with premenopausalTNBC, which is the most deadly form of breast cancer, if anything, thisbias should have decreased the ability to show such associations forwomen with the KRAS-variant.

While prospective studies are ongoing, women with the KRAS-variantappear to clearly be significantly at risk for multiple primary breastcancer. Those at highest risk are women shown to be homozygous for theKRAS-variant, a genotype occurring in ˜3% of the population,significantly more frequent than BRCA mutations (˜0.25%).

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. The scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

INCORPORATION BY REFERENCE

All publications and patent documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if the entire contents of each individual publication orpatent document was incorporated herein.

1.-5. (canceled)
 6. A method for treating cancer in a KRAS-variantsubject, the method comprising: gradually decreasing estrogen exposurein the KRAS-variant subject to reduce the risk of aggressive tumorgrowth.
 7. The method of claim 6, wherein the cancer is breast cancer,ovarian cancer, or non-small cell lung cancer.
 8. The method of claim 6,wherein estrogen exposure is gradually decreased by antagonizingestrogen function.
 9. The method of claim 8, wherein estrogen functionis antagonized by increasing dosage of an estrogen or estrogen receptorantagonist.
 10. The method of claim 9, wherein the estrogen receptorantagonist is tamoxifen, clomifene, femarelle, ormeloxifene, raloxifene,toremifene, lasofoxifene, ospemifene, afimoxifene, arzoxifene,bazedoxifene, or fulvestrant.
 11. The method of claim 9, wherein theestrogen receptor antagonist is anastrozole or fulvestrant.
 12. Themethod of claim 6, wherein the method further comprises detecting asingle nucleotide polymorphism (SNP) at position 4 of the let-7complementary site 6 of KRAS in a patient sample to determine thepresent of the KRAS variant.
 13. A method of predicting an increasedrisk of developing a second, independent breast cancer in a subject,comprising detecting a single nucleotide polymorphism (SNP) at position4 of the let-7 complementary site 6 of KRAS in a patient sample whereinthe presence of said SNP indicates an increased risk of developing asecond, independent cancer in said subject.
 14. The method of claim 13,wherein the second, independent cancer is breast cancer.
 15. The methodof claim 13, wherein the subject is a breast cancer patient.